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Cupins: A New Superfamily ofFunctionally Diverse Proteinsthat Include Germins and PlantStorage ProteinsJim M. Dunwell aa Department of Agricultural Botany, School of PlantSciences, The University of Reading, Reading, RG66AS, UKPublished online: 15 Apr 2013.

To cite this article: Jim M. Dunwell (1998) Cupins: A New Superfamily of FunctionallyDiverse Proteins that Include Germins and Plant Storage Proteins, Biotechnology andGenetic Engineering Reviews, 15:1, 1-32, DOI: 10.1080/02648725.1998.10647950

To link to this article: http://dx.doi.org/10.1080/02648725.1998.10647950

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1Cupins: A New Superfamily ofFunctionally Diverse Proteins tbat IncludeGermins and Plant Storage Proteins

JIM M. DUNWELL

Deparl1nellt ofAgricultural BotanYf School ofPlant Sciences, The University ofReading, l~eading RG6 6AS, UK

Introduction

This review will describe the recent identification of a wheat protein, gennin, whosefunction as an oxalate oxidase was first revealed in the early part of this century butwhich has relnained largely overlooked since that time. Discovery of the sequence ofthis important enzyme (it is widely used in medical diagnosis) has now led to thefurther identification of a wide range of related proteins with a siInilar ~-barrel

structure. This ne\v protein superfanlily - designated 'cupins' - include several smallnlicrobial proteins, and many plant storage proteins that are duplicated versions of theunderlying gennin sequence. Most recently, honlology modelling based on thestructure of phaseolin has been used to generate a 3D structure of germin and it isexpected that this information can be utilized to study the active site of this enzyme andits relatiYes.

Oxalate oxidase

HISTORICAL OVERVIEW

An enzyme with oxalate oxidase (ox-ox) activity, that converts oxalic acid to carbondioxide and hydrogen peroxide, \vas first reported by Zaleski and Reinhard (1912)from studies ofpowdered wheat grains. Remarkably, it was Inore than 80 years beforethe identity of this enzyme was confirmed in cereals (Lane et al., 1993; see below).After the first report, the enzyme was studied in both wheat flour and bran by Palladinand Lovchinovskaya (1916) and in 1928, Zaleski and Kukharkova concluded that thethis enzyme should be considered as a dehydrogenase able to use only oxygen as ahydrogen acceptor. Meanwhile, Staehelin (1919) had reported a similar enzyme fromleaves of several higher plants, including some known to contain oxalic acid, such asRunzex, RlteUl1l. and Spinacia. However, the most spectacular case of oxalic acid

Biotcclmol(J8Y alief G~l1elic: l!.·lIgineeri"g Rev;ew.f - Vol. 15. April 19980264-8725/98/15fl-32 $20.00 + $0.00 @ Intercept Ltd, P.O. Box 716. Andover. Bampshirc SPIO IYG, UK

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degradation was found in Jnosses - a source first discovered by Houget etal. (1927)in HYPIlUI1Z triquetrunl, //. (.'upress{t"orJne, 7'hudiulll abietiluull <Old Poly/ric/nun.jUlliperllln. In subsequent work, Franke and Hasse (1937) characterized the enzylnefrollt Hyaloc0J11illI11Unlbrlltlll1l, and noted a rernarkable thefillostability (cf. belo\v for\vheat germin). This \vas followed by Datta and Meeuse (1955) \vho designated thetTIOSS enzynle as a flavo-protein.

A1110ngst the cereals, ox-ox activity has probably been best characterized at adeveloplucntal level in sorghun1, although in contrast to wheat, barley and rice (seebelow) no protein sequence infonnation is available fr0I11lhis species. In the first studyon sorghunl, Pundir and Nath (1984) lIsed the 15 000 g supernatant froln lO-day-oldleaves and found enzynle activity with a pH optinlunl of5 and an absolute requirelnentfor ITIctal ions, particularly iron. 'fhis finding seelns to be contradicted by theconclusion frolll subsequent studies (Pundir, 1991; Kuchhal et £1/., 1993) that copperis the criticullnetal ion. This latter conclusion \vas supported by a study on roots fromthe same nUl.terial (Pundir and Kuehnal, 1989) vslhich sho\\/ed an involve.lnent ofcopper in pr01110ling enzynle activity.

Sinlilar studies on barley ox-ox have been conducted by Chiriboga (1966) andSugiuraet al. (1979). The Jnost recent, und Inost thorough, study of the barley enzymeis that reported by Kotsira and Clonis ( 1997) who purified ox-ox to homogeneity bylhennal treatlnenl (60°C for 10 Inin)t followed by two affinity chromatography steps.It was concluded that the enzyme is activated by I mM each ofCa2+ and Pb2

+, and thatsOlne of the cysteines nlay act to preserve the catalytic activily by nlaintaining theintegrity of the pentameric asselnbly via disulphide bond formation. Optilnization ofthe assay for ox-ox activity has been reported by Zhang et al. (1996) \vho showed a10-IOO-fold increase in sensitivity if 600/0 ethanol is included in the assay nlixture.

COMtv1ERClAL SIGNIFIC,\NCE OF OXALATE OXIDASE

The cOlnlnercial significance of this enzynle cOlnes hrrgely from its widespread use·inkits to assay oxalate levels in blood plasma and urine. Indeed, regular assessment ofsuch levels is required for control of hyperoxaiuria, whether it be the rare geneticcondition which leads to deposition of calcium oxalate throughout the body (De Pauwand TOllssaint, 1996), or the much more frequent incidence of urolithiasis - theformation of kidney or bladder stones (Runl} et 1I1., J997).

By far the rnost common source of enzyme used in these kits (e~g. Signla orBoehringer MannheiIn) is barley roots. Although the assays are siJnple and quick,continuous efforts are being nlade to inlprove the accuracy still further. Such studiesinclude ilnmobilization of the enzyme either on glass beads (Hansen et aI., 1994), onAF-l'resyl ~royopearl650gel (Yamato et al., 1994)t on a pre-activated nylon mem­brane (Saka Aillini and YaHoo, 1994), or by addition of ethanol to the assay mixture{Zhang e/ (II., 1996). The products can be quantified by colorimetric (Petrarulo et aI.,1994), amperometric (Saka Alnini and Valian, 1994; Yamato et al., 1994) or chelni-luminescence (Balion and Thibert, 1994) methods. SiInilar reports on the enzymeextracted from beet stenlS (Obzansky and Richardson, 1983,1984; Varalakshmi et ill.,1995) include one on immobil ization on concanavalin A (Varalakshmi and Richardson,1992), a method clailned to eliminate the significant inhibition produced by azide,nitrate (Meeuse and Canlpbell, 1959) and glycolate.

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AItcrnative sources ofenzyme include IllQSS (Lakeret til., 1980) and especially bananapeel- the preferred source in tropical countries (Inamdaret (ll.~ 1986,1989; Lalhika etal.,I995a, b). Degradation of the oxalate found in banaoasin situ had been known for tnanyyears previously (Wylllan and Pahner, 1964: Shimoka'W·a et (II .. 1972).

<>XALATE AND HUMAN METABOLISM

Although a high level of oxalate in plaslna is usually considered to be a pathologicalcondition (see previous section), retnarkably high values in blood cells~ \vith aJ11axilTIUlll of 291 0 J1nlolcsll~ \vere reported by Albrechll~llll. (1994). These authorssuggest that oxalate 1l1ay not silnply be an end product ofInelaboHstll, but rather it rnayhave an inlportant physiological function:- perhaps by providing the hydrogen perox­ide generated by phagocylcs during the in fJarntnatory process.

OXALATE DEGRADATION AND GENE THERAPY

As described briefly above, primary hyperoxaluria is a rare, and sometimes fatal..heredilary disease (De Pauw and Toussaint, 1996). There are known to be two fonlls,type I \vhich is due to a deficiency of the liver-specific peroxisolnal enzyInealanine:glyoxylate anlinotransferase/serine pyruvate aJnino-transferase, and lype 2which concerns glyoxylate reductase/D-glycerate dehydrogenase, a cytosolic cnzynlepresent in leucocytes and hepatocytes. Several gene therapy approaches are beingconsidered to alleviate these conditions. They range from introduction of the appro­priate \vild-lype version of the endogenous gene to expression ofan alternative oxalatedegrading enzylue. The latter approach was presaged by (he observation that ratsinlplanted with dialysis Inenlbrane capsules containing irnlnobilized ox-ox fr()ln

banana (see above) could effectively Inetabolize intraperitoneally injected [14C]oxalate as weB as its precursor, [1 4C] glyoxyJate (RaghavHn and Tarachand, 1986) (seealso Batich and Vaghefi, 1994).

Although their eventual anlbition was to clone the fungal oxalate decarboxylasegene for use in gene therapy (finlayson and Peck? 1988: Sidhu el al., )997) Lunget al.( 1991,1994) did succeed in cloning a bacteri~d equivalent, the oxalyl-eoA decarboxy­lase froln()xalobacter.f'orI11igelles, an oxalate degrading anaerobic bacteriulll found inthe InamJnalian inlestine (Baetz and Allison~ 1989; Cornick and Allison, ]996;Kuhner et al., 1996). This approach has recently been superseded by the cloning andpatenting of the hlunan version of this enzyrne (Colelnan et (II., 1997) and thepublication ofseveral related expressed sequence tags (ES"fs) frolll hUlllan (gil2238232,gil 1727767, gil 1722162), Tllouse (giI2192248):- Caenorhabditis (gill 0448 14) and yeast(gil602387) sources. It is likely that the recent advances in understanding the sequenceand structure of ox.alate degrading enzylnes (see below) \vill help in this area ofresearch, which in many \vays is equivalent to the work on transgenic plants alsodescribed belo\v.

OXALATE DEGRADATION AND BIOREMEDIATl{)N

An interesting, non-Jnedical application of oxa]ate degradation is provided by theexample (Anon., 1992; MOl1on e1 al., 1994) of using PSeUdOl1l0naS oXlilaticus

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4 J.M. DUNWELL

(Chandra and Shethna, 1975)~ a bacterium found in rhubarb patches, to digest sodiurnoxalate, a hazardous byproduct of the Bayer process (Farquharson el al., 1995)utilized in altnninium sJ11elters. The specific bacterial enzynle responsible for thisdegradation has nOl been isolated to date.

Gerolins and germin..like proteins from plants

GERM IN IN CEREALS

DiscoVel)' (~roxalate oxidase activity

The onset of growth in germinated wheat ernbryos is accompanied by the productionof a soluble, pepsin-resistant homopentanleric glycoprotein (oligonler approx. 125kD) that was initially called'g' and then named ~gernlin' (Grzelczak and Lane, 1984;McCubbin et al., 1987). In subsequent studies, the sequence of two very sinlilargermin genes designated gf~2.8 and gJ~3.8 were obtained by Dralewka-Kos el ill.( 1989) and Lane et Cli. (1991). The isoforms of germin are discrete temporal markersof wheat enlbryo development; wall-associaled gerlllin accounts for abollt 400/0 oftotal genl1in in gerlllinating wheat enlbryos, with the appearance of germin in theapoplast being the Inost conspicuous germination-related change in the distribution ofcell-wall proteins (Lane el €II., 1986., 1992). Although these studies revealed IlluchaboLlt details of the germin protein and its expression, its funClion rernained unkno\vnfor several years. Only when the sequence of barley ox-ox became available was itrevealed that genllin gf-2.8 was in fact a protein with ox-ox activity (Laneet al.,1993;Duma~el at., 1993) and was therefore presumably the enzyme first identified sonle 80years previously (Zaleski and Reinhard, 1912). It is thought that the closely related gf­3.8 is monolneric and does not exhibit ox-ox activity. This difference creates apotential probleln of n0l11enclature, in that silnilar genes encode proteins with quitedifferent properties, and therefore functions. It is proposed to restrict the term gernlinto the cereal genes and to stress that the gene products may have a range of function.

Gerl11ins ill cereals

It was discovered at an early stage in the study of gennin that an immunologicallysinlilar protein could be detected in all cereals tested, i.e. in the slenlS of barley't oat!tand rye (Grzelczak ellll., 1985) and in maize and rice (Lane et aI., (991). However,no such protein was detected in pea, lettuce or radish, nor in the moss Tor/ula ruralis,although as the authors state, 'such findings do not bear, definitively, on the occur­rence of proteins related to germin' (see belo\v).

Of all cereals tested to date, the largest number ofgerrnin-like gene sequences havebeen found in rice. These comprise one 20 AA fragment (giI348652) from a 25 kDprotein and 20 ESTs. The EST sequences are from 8-day-old shoots, either green oretiolated, or froln the panicle at the ripening stage (Table 1). Translation and manualediting of obvious sequencing errors reveals a possible total of eight distinct proteins\vhich can be divided into three classes (Dunwell, unpublished results). The first ismost silnilar (approx. 700/0) to the Prunus ABP 20 protein (giI1916809), the second isrelated to the barley X93171 protein, and the third is similar to the Arabidopsis GLP

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Table 1. List of Rice ESTs which encode gc.rmin~

1234;6789

10II121314151617181920

dbESTld

71171716047]9167215172J547232772360724157557675954

144047144301144848145340146125146169146752146941726312726835

Library

shootshootshootShOOl

sh()otshootshoo.shootshootshootgreen shootgreen shootgreen shootgreen shootgreen shootgreen shootgreen ~ho()t

green shOOIpanicle at ripening stagepanicle ell ripening stage

2b germin-like protein. Interestingly, there are no examples closely related to thewheat gennin or barley root ox-ox.

Pallerns o..fexpression

In 3-day..oJd barley seedlings, oxalate oxidase is localized in the epidermal cells oftheInature region of the primary roots, and in the coleorhiza, whereas after 10 days ofgrowth it is only detected in the coleorhiza (Dumas et al., 1995).

Function

Deve!opllIelZl. Two features of wheat germin have been linked lO specific roles inplant development (Lane, 1991), particularly in the modification of the extracellularmatrix (Lane, 1994). First, the tight adhesion of germin to lhe highly substitutedglucuronogalactoarabinoxylans (GGAX) suggests a possible role in incorporation ofthese compounds into the growing wall. Secondly, the generation of H

20

2is required

for peroxidase-mediated reactions such as lignification, and the cross-linking ofcoumarates, extensins and ferulates to cell wall hemicelluloses and pectins. In directcontrast to the first role, these reactions would stop the process of wall extension. Thepresence ofseveral auxin responsive elements in the promoter ofgenningt:2.8 (Bemaand Bernier, 1997) is additional circumstantial evidence for a link between cell wallpH and gennin activity (Lane et tIl., 1992).

Plant pathogen interactions. In a preliminary study of defence-related gene

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expression in barley, Wei (1994) identified a eDNA clone (gil I070358~ X93171) \vithapproximately 45% identity to barley root oxalate oxidase. Il is now known to be nlostsimilar (900/0) in sequence to a reconstructed protein encoded by the fusion of t\VO riceESTs~ gil1632028 and gi11632551. Subsequently. there have been two particularlysignificant publications (Dulnas et (II., 1995; Zhang et al., 1995) describing theinvolvement of ox-ox in the response of barley plants to powdery nlildew infections.In the former study, it \vas sho\vn by immunoblots and direct assays that the enzyolcwas induced in leaves between three and five days after inoculation, and that it waslocalized 1l1ainly along the vascular bundles. There was no induction by wounding.T"he latter investigation denlonstrated a ITIuch lTIOre rapid response, with colorillletricassays and activity blots showing a detectable increase after 24 h and a lO-foldincrease after 48 h. These increases appeared 1-3 days earlier than PR-I acculllulationand it is suggested that the activity results frolIl de IlOVO protein synthesis. thoughpathogen-induced enzynle activation is also possible. It is not known whether theH.,O., generated by the enzymatic process is used as a signal for the induction of otherc~mponents of the defence response~ for lignification, or directly to inhibit pathogendevelopnlent (Thordahl-Christensen et al., 1997; Wojtaszek. 1997).

Silnilar results \vere reported by Hurkman and Tanaka (1996b) for wheat leavesinoculated with the same pathogen.

Salt stress. There have been several, related studies on the effects of salt stress on theexpression of gernlin genes in barley (HordeLl11"l vulgare L. cv eM 72) seedlings(Hurkmaner lIf., 1989,1991,1994; Hurkman and Tanaka, I996a). In control seedlings,germin mRNA levels (as estimated by expression of sequence P4585 1) are regulateddeveloplnentally, with the highest amount in roots and the lowest in shoots. Theselevels are maximal 2 days after inhibition, before declining. In contrast, for seedlingsgrown in the presence of 200 InM salt, the levels remain high for an additional day.Expression patterns in salt stressed roots are 1110re complicated, with levels rising onlytransiently when 6-day-old roots are exposed to 200 mM Nael (Hurknlan and Tanaka,1996a).

OXALATE CONTENT OF CEREALS

In the light ofthe discoveries that a predoJninant gernlination-related protein in cerealshas ox-ox activity and that this protein has several close relatives, it is pertinent toconsider the state ofknowledge of oxalate in cereals, particularly since this compoundhas never been considered to have any significance in this group of plants. For ageneral review ofoxalate in crop plants, the reader is referred to the survey by Libertand Franceschi (1987).

There have been few efforts to localize oxalate in cereal planls.ln an interesting andpossibly unique histological study, Prankerd (1920) reported the presence of calciumoxalate crystals in the nodes of the wheat plant. It was inferred that these crystalsplayed a role in the detection ofgravity and they \vere estimated to travel from one sideof the cell to the other in under five minutes. Much later, Wagner (1981) reported thatoxalic acid in barley is mostly localized within the vacuoles.

Attempts to quantify the levels of oxalate in cereals are also scarce. Nelson andHasselbring (1931) eSlitnated the amount in wheat plants to be 0.020/0 on a fresh

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weighl basis (0.11 % by dry weight), and values of 0.019% for barley, 0.029% for11laize, 0.04% for oats, and 0.048% for rye were calculated by Nelson and Mottern(1931). In a Inore detailed physiological study on Inaize, Wadleigh and Shive (1939)measured values of oxalic acid on a dry weight basis, ranging from 0.062% for plantsgrown at pl-l 3.0 to 0.178% for plants at pH 8.0. However, in the same year, Olsen(1939) could not detect oxalate in leaves of barley and maize, whereas in a study ofnutrient conditions on the growth of oats, Pepkowitz et al. (1941) showed the highestoxalate levels in those plants supplied with additional nitrate. l~he mosl relevant ofthestudies fronl that period is probably that of Andrews and Viser (195 I) who lneasuredthe oxalate content of wheat elnbryos and found a value of about 1 ~lg/elllbryo. Anyoverall conclusion froln these studies must be qualified by the proviso that theaccuracy of the values cannot be assessed according to modern standards, butnevertheless they do show that low levels of oxalate are recorded in most cereals, andthat levels in whole plants are modified by a range of environmental variables.

A specific defence role for oxalic acid in rice was suggested by the findings ofYoshihara et al. (1990). These authors showed conclusively that this acid could beisolated frOlll the leaf sheath and that it acted as a potent inhibitor of sucking by thebrown planthopper.

TRANSC]ENIC PLANTS EXPRESSI.NG OXALATE OXIDASE

To date, the only practical application of germins in transgenic crops is based theiractivity as an OX-oX, and its potential use as an antifungal agent.

Oxalic acid is produced by several plant pathogenic fungi (Loewuset al.~ 1995) andis considered to play a major role in the pathogenicity of Sclerotinia sclerotioruln(Noyes and Hancock, 1981; Rowe, 1993), a particularly important pathogen ofsunflower (Masirevic and OuIya., 1992; Sackston, 1992) and many other dicot crops.Evidence for the role ofoxalic acid comes from studies on mutant strains ofthe fungus\vhich are deficient in oxalate production and also avirulent. Revertants for acidproduction regain their virulent nature (Godoy et al., 1990).

The proposed mode of action for oxalic acid in producing disease symptoms is firstthe cheJation of calcium from the pectate fraction of the xylem and associated pitvessels. secondly the entry of air leading to xylelTI embolisms and wilting (Sperry andTyree, 1988), and finally the reduction in pH which stimulates the activity of thefungal enzymes such as polygalacturonase, methyl esterase and cellulase (Marcianoetal., 1983).

Analysis of the role of oxalic acid, as described above, led to the strategy ofintroducing oxalate degrading enzymes into plants as a means of protecting themagainst the fungal toxin (Thompson et al., I995a,b). This transgenic strategy, whichhas some paralleJs with the work on human gene therapy mentioned above, hasutilized both available genes., the ox-ox froJn barley or wheat (see above) and thefungal oxalate decarboxylase (see below). Field studies are Inost advanced on transgenicoilseed rape material containing the fonner gene (J.M. Dunwell and C. Thompson,unpublished results). When grown in field conditions containing a high level ofScler()tin.ia, the transgenic plants show a significant level of protection against thepathogen. It is not yet known whether this is sufficient for commercial exploitation.

A sitnilar transgenic approach, utilising bacterial genes for oxalate degradation (cf.

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Koyanla, 1988), has been suggested in a study ofSclerotinia infection ofArabillo!Jsis(Dickman and Mitra~ 1992).

In addition to their value in applied studies described in the previous sections,transgenic plants have also been used in a series ofmore fundamental studies such as thatin which the wheal gf-2.8 gennin gene was introduced either as the intact gene, or aspromoter-GUS fusions, into transgenic tobacco (Berna ~UldBernier, 1997). Heterologousgene expression was lTIonitoredill vitro and ill vivo by GUS orox-ox activity, and shownto occur in developing seeds and in seedlings. This transcriplion was slimulated by auxin,probably o\ving to the presence ofseveral auxin-responsive elements (e.g. TGTCCCA1"and TGTCTC) in the promoter sequence. Interestingly (in view of the gemlin/vicilinconnection; see below), this sequence also contains nine 'legumin boxes' scatteredbetween -1530 and -1142 upstream. Analysis of the transgenic protein itself revealedtwo enzylnalically active isofonns, corresponding to the wheat germins G and G' whichdiffer by the presence of antennary N-acetylglucosamines (Jaikaran et al., 1990). Thisanalysis suggests that the post-translational modifications of the protein (oligomerassembly, stability, glycosylation) are very sinlilar, if not identical, in the hOfDOJOgousand heterologous hosts.

GERMIN-LIKE PROTEINS (GLPs)

This group of proteins includes all those plant proteins closely related to the cerealgennins. It is, therefore, a somewhat arbitrary designation.

Gyl1l1Z0Spernzs

Pinus caribea. In a study of extracellular glycoproteins secreted by embryogeniccultures of the Caribbean pine (Pinus caribea Morelet var }ro1Zdll.rensis), Domon el (II.(1995) identified three proteins with some similarity to germins. This sinlilarity wasbased on N-tenninal sequence from one protein, and cross reactivity with antibodiesraised to the nonglycosylated wheat monomer as weJl as to the glycosylated barleypentameric protein. Interestingly, these proteins were absent from the control oon­embryogenic cultures and they therefore represent the first markers of somaticembryogenesis in this species.

Pinus radiatll. A study of a cDNA library made from somatic embryogenic tissuehas recently revealed four ESTs (gill 839644, 1839645, 1839658, and 1839659) withsignificant similarity to germins (S.L. Bishop..Hurley et ill. unpublished results);Northern blotting using 5 J.tg RNA showed no expression in roots, stems, needles andcallus. Manual editing (J.M. Dunwell, unpublished results) on a combination of thetranslated products from these ESTs suggest that they probably represent a genewhose protein product is given in Table 2. This sequence is most similar (44%identical, 62% similar) to the Arabidopsis germin-like protein GLP8 (giIl755192).

Dieot specie,')

Arabidopsis. In Arabidopsis, there are a total of 27 protein sequences (gill?55152-

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Table 2. Putative sequence or gcrlnin-likc proteins from Pinus rlldi(.lta (a) and Strawberry (b). Thesesequences have been produced by translation and manual editing of various ESTs (J.M. Dunwcll.unpuhlished results~ sec text for details)

(a)

DPEAI.QDR.'VADTKSS(FMNGRF{lNPfv1F~PEHRv11SALRKAGNLSANPlGFSVIVTIl.'ANMPGI..NI1.a....c;MG

RlDMAPGSAIPPHJHFRGSETIXVVRGALNVGFVDTSNRLXXHKLvrGDVFVFPKGAVHYl.QNIGKITAAVSAFNSQNPGTAIVSLAfFASNPAIPYEVLSKAFAISVQEVSQIRKSFGGT

(ll)~i'AAMQDf~VADYAAfQSPAGYACKDPAK~"VFSGLRVAGNISSINKIGI.SMFA VNFPGL.NGLGVALVRADFAVGGVVPlHSHRDAT8...V(LVER1WAGRAXNNKAI..cFXSA~GR1PALXYA~

FSSANPGVQTLEXTLFXNELPTEIIXNSTLLEKXXIXKL

1755192, gif1934726-t934730, gill107491, gill 592672, giI2239042), two BAC se­quences (giI2091318,giJ2092889 which encode in the negative slrand a protein verysimilar to gil1755165), and 31 E~Ts in the databases (see also Delseny et al., 1994;Hofte et al... 1993) and these can themselves be sorted into a number of classes (J.M.Dunwell, unpublished results). The proteins seem to comprise 12 distinct sequences,reduced to 8 if a nlore conservative estimate is made.

Brassica. The only full length sequence of a Brassica gennin..like gene in theGenBank database is accession U2 I742 produced from 5-day-old etiolated seedlingsof Brassica llapus cv Samourai. Its closest neighbour (87% identical, 920/0 similar) isan Arabidopsis ~gernlin' GLP 3 (U75 189). However., three olher partial sequenceshave been isolated fromB. napus by the useofPCR based methods (A. WaIkerand I.J.Evans, unpublished results) and these are described in Table 3. In terms of similarity,clones 1and 2 are most similar (84%) to a section of Arabidopsis GLP6 (U75194) andclone 3 shows close similarity (730/0) to a section from the barley germin X93171.Other ESTs are also known, gblH07760 froID B. napus and gbllA6495 from B.clll1rpestris. With the insertion of an additional ~t' at position 174, the fonner ESTencodes a sequence identical, with the exception of one amino acid, to the B. llapussequence U21742 (1.M. Dunwell, unpublished results).

Si1lapis. As part of an attempt to analyse rhythmic phenomena in the long day plantSinapis alba, Heintzen et 01. <. 1994) searched for mRNAs whose concentration variedwith the time ofday. ''[hey identified a gennin-like transcript that underwent circadianoscillations in light/dark cycles with maxima at around 8-9pm and minima at aroundSam. At a cellular level, the transcripts were expressed in the epidennis and spongyparenchyma of young leaves, and in distinct regions of the epidennis and cortex ofstems and petioles. The protein itself (giI683488) was associated with the primary cellwalls.

Pharbitis. In a very similar study to that on Sinapis described above t Ono et at(1993) llsed the short day plantPharbitis nil in an examination of floral induction" Theconvenience of this species is clearly demonstrated by the fact that a single 16-hourdark treatment is sufficient to induce flowering. During the second half of theinductive dark period the level of a specific 22 kD protein (isoelectric point 7.5)

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Table 3. Germin-like sequences fronl BrassicllluZpllS (A. Wulker and 1.1. Evans. unpublished results)

Clone ICXJICAGltI..lXXD:lXAI'P:IOCA:n:OX.xnx:GACT(]AGKIITlOOI1UIffi\ACAAOOAACCrIOCI1UfAOOarfIGTGIl:rrCAAACJ::AAGA1lJAGAAa::cn:crrno:JCCCOClCAATGITGGIGA"IUfA1TIUIUfI1CCAGAAGGCrrccrccATI1'TCAACICAAGGCATL"LGGGCfAGAGCGGCGCA<X:GCGG1OOACCICCCAG

Truns)ation:RQSPPHMHPI~IEILWQQG'lU.VGFVSSNQDENRLFAKrIlNVGDVFVFPEGFLHFQLKASGIFRRTAVELP

Clone 2caXX:O:)XIJ\1OCATCCCCo.:rr:CA(~roAGA1CI1UATrG1(cAACAClJGAACCI1OCf1U1}\GGGl"I1UIt:1C

TfCAAACC'AAGAlUGAAACCGR:TM1'COrAAAACGCIQ\AlUITOOIOA1UI"ATI1GTGJTI'CCAGAAGGCCTCATCCA1""I1~rCAACTAAOGCATCCAOOGCTAGAGCGGCGCACCGCGGTGGAGCTCC

Trnnslation:PPHMHPRATEILIVQQG~11..LVGFVSSNQDGNRLFAK'n~NVGDVFVFPEGLIHFQLRHPGLERRTAVEL

Clone 3Translation (by rn"nual editing):PPHMHPRAREII.:IVLEGILRVGfVrSNPNNRFTIKVLKKGDVFVFPVGLVIFQSNVGNGNAVAIAALSSQNPGAlTIANXVFG

increased significantly. This protein (gil662292) was subsequently identified (Ono el

al., 1996) as a germin-like protein whose nlRNA was detected particularly in theyoung expanded cotyledon and leaf. The level peaked about 10 hours front thebeginning of the dark period, in contrast to Sinapis where the peak for the relatedprotein occurs about 12 hours after the start of the light period (Heintzen et at.,1994).

Interestingly, this Pharbitis protein was tested and had no ox-ox activity.

MeSe111bJyalZthe111UI1t Mesentb,yalltheJrIlI11l. c/:vs(allbuull is a facultative halophyteand has been widely used in studies of biochelnical changes and gene expressionduring water stress (Bohnert et a/., 1988). In a detailed biochemical study, Adalns etal. (1992) found an increase in oxalate content in the bladder cells fronl <1 roM up to106 mM as salt levels were increased fronl 1mM to 5 mM. 'This finding may be linkedto the results obtained from a differential screening of a eDNA library produced froIDroots subject to 6 hours of salt stress (500 rnM) (Michalowski and Bohnert, 1992).These authors identified a significant decline in the transcript of a germin-like protein(gill 169942). In a subsequent study of changes in transcript levels for several genes,Andolfatto et al. (1994) found that a reduction of 'germin' message in roots after30 hours of salt stress (400 mM) but an increase afler 120 hours.

Detailed comparison of the protein sequence from this species \vith other gennin­like proteins reveals an obvious loss in similarity towards the C-terminus at amino acidposition 197. Before that residue, there is approximately 750/0 sinlilarity to Arabidopsisgermin U75192. Beyond that residue there is no significant sitnilarily to any othergermin. However, if an additional nucleotide is inserted at that point (position 609 ingil167257), then the similarity is regained (J.M. Dunwell, unpublished results). Thissuggests either a sequencing error which introduced the effect of a frante shiftmutation, or that indeed such a lnutation occurred during the evolution of this protein.There is no evidence available for distinguishing between these alternatives.

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CUllins 11

Prullus. "[here have been two recent database submissions ofgermin-like sequences(U81 I62,U81163) fronl peach (PrU1JllS pel:ric(l cv Akatsuki). The authors of this !\tudydescribe these sequences as auxin-binding proteins (ABPI9/20) (see below fordiscussion of ABPs), but this is an obvious JniSn0l11er since their closest relative in thepublic database is an Arabidopsis 'germin' sequence GLP 2B (U75193). However,they are even Inore similar to a previously unpublished sequence from strawberry (seebelow).. another member of the Rosaceae.

Strllll'ber/J. As part of a large-scale EST project on gene expression in strawberryfruit, a germin-like sequence has been identified recently (A. Van Tunen, personalcOlntnunication) (see °Tllble 2). Not surprisingly, it is tnost shnilar (54% identical, 72%silnilar) to (he sequence from peach mentioned in the previous paragraph.

Legul1zes. Although Staehelin (1919) reported the oxidation of oxalic acid by flourproduced from a range of legulnes, nanlely Lupilllls albus, Phaseolus vulgaris,Glycine soya, Vicia .{aba, and PiSlllll slitivUl1l, the first evidence for the presence of agcnnin-like protein in any legumeop caIne froIII the work ofSwartet (II. (1994) on a cellwaJ I protein froIII pea roots. This protein was hnplicaled in the attachlnent ofRhizobiulll and Agro/)acteriull1 bacteria to the plant cell wall, a process mediated bythe bacterial protein rhizadhesin. Analysis showed the putative receptor to be aglycoprotein with an isoelectric point of 6.4, a molecular mass of 32 kD before~ and29 kD after glycosidase treatment, and to have an N-terminal sequence ofADADALQDLCVADYASVILVNGFASKPLI (with some doubt over the last threeresidues). Use of this sequence in a BlastP search reveals the closest relative (69%identity) to be an Arabidopsis germin (giI1934730) and two ESTs (W43342~

AA042591). It lnay be of interest to note that the most successful extraction methodfor rellloving the receptor from the cell wall involved the use of an oxalale/oxalic acidmixture.

More recently .. this evidence from pea has been supplemented by the directnleasurelnent of oxalate oxidase activity in nodules of faba beans (Vicill fllba L.)(Trinchant and Rigaud, 1996). These authors, who had previously detected high levels(70 InM) of oxalate in nodules (Trinchant et al., 1994), have now demonstrated thatapplication of a water stress increases the level of oxalate oxidase activity fourfold,and reduces the level of oxalate by 55%. The enzyme itself is characterized by anoptimal activity at pH 8, in contrast to the cereal enzyme referred to above, which hasan optimum at pH 3. It is suggested that the oxalate present at high levels in the nodulescould playa role as a cOlnplementary substrate for bacteroids, and as a means to slowdown the decline in nitrogen fixation induced by water restricted conditions.

In the light of these findings of high levels of oxalate in nodules it is somewhatsurprising that legumes show susceptibility to Sclero/il1ia (see above).

MAPPING INFORMATION FOR GERMINS AND GERMIN-LIKE PROTEINS

There have been two studies to locate the position of the gennins on the cereal genomemap. In the first, quoted in Lane et al, (1991) it was reported that a germin eDNA usedas a probe mapped primarily to chromosomes 4A (--5 copies), 4B (-3 copies) and 4D(-9 copies) in hexaploid wheat. In the second, Dubcovsky et al. (1995) used the barley

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clone pWJHGennin (Hurknlan et al.., 1994) on a mapping population of TriticUl1lI1UJIlOCOCCU11Z and found one pair of fragments mapped to the centromeric region ofchronloSOlne 2Am (arID location unknown) and another locus was Inapped to chrOIDO­

sorTIe 4Anl, The position of this locus is similar to thal previously reported by Lagudahet al. (199 I) for Triticunz tauschii and Devos et til. (1995) for wheat chrOInOS01l1eS 48and 4D. In an unpublished study (Gale el a/.) quoted in Berna and Bernier (1997), ithas been shown that the promoter of the gf-2.8 gene is largely confined to the D

genolne of wheat; it hybridizes to four genomic fragments from this genome, one fr0l11the A genome and appears to be absent from the B genonle.

The only information from rice is unpublished data provided by Dr Ilkka Havukkalu(Rice Genome Research Program) \vho reports thal the rice clone S1623 (=039942)Inaps to the top tenninus of chromosome 4 (Koike et {II., 1997), at the same locationas Inarker C445 on the map published by Kunltaet al. (1994). It is likely that there isonly one location for this clone in the genome,

There is no published infonnation on the chrofilosomallocation of any of the dicotGLPs, although it can be expected that the Arabidopsis genes will be placed on theInap in the near future.

Microbial and animal proteins with sequence similarity to germins

SPHERULINS AND DESIGNATION OF THE 'GERMIN BOX'

The first proteins found to have any significant sequence similarity to the wheatgermins were the spherulins" proteins expressed in the sHine Inould Physaru111IJolycephllhtl1l during the process of plasmodial encystlnent or spherulation (Bernieret al., 1987). Plasnl0dia will encyst to fonn spherules when subjected, in the dark, todehydration, cooling, acidic pH, sublethal concentrations of heavy Inetals, highconcentrations of some carbohydrates, or starvation. These spherules are hard-\valledoJigonucleated subunits produced by cleavage of the plasmodia, and they can relnainviable for lTIany years.

At the alnino acid level there is 44% sinlilarity in sequence bet\veen germin gf-2.8and spherulin 2b (Laneet lIl., 1991), a value that increases to about 60% for the centralcore sequence. Near the centre of this conserved core is the decapeptide sequencePH(Iff)HPRAI'EI, a region designated as the 'germin box'. In the sections below, itwill be denlonstrated that this Inotif is conserved across a Inuch \vider range ofproteinsand is part of the putative active site of the cereal ox-ox enzynles (Gane et al. t 1998).It should be noted however that the spherulins do not show ox-ox activity and theirbiochenlical function remains unknown.

IDENTIFICATION OF TWO HISTIDINE-CONTAINING MOTIFS

In a search for possible germin progenitors, BLAST searches \vere conducted onvarious ~gemlin' and 'gerolin-like' sequences in the GenBank database. Subsequentanalyses (J.M. Dunwell, unpublished results) using the BLOCKS (Henikoff andHenikoff, 1991), MEME (Bailey and Elkan, 1994) and PROSITE programmesshowed first that only certain residues within the 'germin box' are absolutely con­served across all proteins, and secondly that other regions of the protein are equally

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Cupins 13

conserved (Dunwell and Gane, 1998). Specifically, it was shown that the ~gennin box'is part of a longer 20/21 AA motif, G(X)5HXH(X)IIG. This sequence is part of thePRINTS Inotif 5GERMINl' and is followed (usually after 15 residues) by a secondmotifof 16 AAs, G(X)sP(X)..H(X)3N (part of PRINTS motif 'GERMIN2'). Using thebean storage protein phaseolin (Lawrence e/ al., 1994) as a reference, these twohistidine-containing motifs are part of the J3-strands designated C/O and alii withinthe two ~-barrel elelnents. Considering the first motif, the two flanking glycines areequivalent to the strictly conserved Gly24

t) and Gly2(l9 which assist in the fonnation of

the shorl interstrand loops B-C and D-E within the C-terminal ~-barrel of phaseolin.Similarly, the second motif contains a proline equivalent to conserved ProJ03 which ispart of the interstrand loop between strands G and H. The variable spacing betweenmotifs is equivalent to the insertions (varying between 7 and 25 residues) tolerated inlhe E-F loop (see Gane et (II., 1997).

CUPINS

The recent sequence analysis reported by DunweJl and Galle (1998) identified a seriesof protein falniIies (rnany bacterial) which contain the two motifs described in theprevious paragraph. These families are considered below and have been divided intotwo categories. The first includes the proteins having a single 5germin-type' domain,and the second include those with a duplication of this domain. Amongst the list ofsingle domain proteins are several that are much slnaUer than the germins, with themost extreme being only 77 re,sidues in length. These 'protogermins' have only a core)3-barrel structure (as judged by homology analysis), with a minimal intennotifspacing and without any terminal a-helices. It is proposed to designate them as~cupins' (frolu the Latin tenn 'cupa' for a slnall barrel or cask). Whilst it is acknowl­edged that enzyme Ilonlenclature is usually based on function, in this case the relatednature ofthe proteins (many with no known function) has been detennined on the basisof sequence/structure. Using this nomenclature, the 'germins' and 'gennin-like'proteins (mean length c. 200 AA) may be considered as 'long cupins' 1 and the oxalatedecarboxylase and other two-domain proteins (see below) as 'bi-cupiDs'.

ONE·DOMAIN PROTEINS

Auxin-binding jJroteins (ABPs)

These proteins are widespread in the plant kingdom, and are thought to aCl as areceptor for IAA, and thereby control a range of plant growth responses (Millner,1995; Napier, 1995; Napier and Venis, 1995; Venis and Napier, 1995). Amongstseveral descriptions of such proteins from both monocots (Palme et al., 1992; Schwobet aI., 1993) and dicots (Choi, 1996; Leblanc et al., 1.997; Lazarus and Macdonald,1996; Shimomura et al., 1993) is one on the isolation of a presumed ABP from peachshoot apices (Ohtniya et at., 1993). However, this protein binds 2,4-D with lowaffinity (Kd=40JlM), it has even lower affinity to IAA and NAA~and it does not cross­react with antibodies to maize ABPI (Fellner et al., 1996). As Jones (1994) states, itsfunction remains unknown. Recently, the sequences oftwo peach proteins gil1916807and gil 1.916809 (Accessions U81162,U81163), described as ABPs, were submitted to

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the GenBank database (A. Ohmiya et al., unpublished results). As mentioned above,this is presunlably a nlisnomer, since they show much closer sequence sitnilarity togennins than to functional ABPs (see details above). This misinterpretation can nowbe explained by the fact that ABPs belong to the one-domain cupin superfamily(Dunwell and Gane, 1998).

These authors recognized a consistent similarity between the sequence ofABPs andthat of germins, particularly in the central core sequence of three ABP exons thatinclude the ~gernlinbox' (motif 1). Within the ABPs, this 1110tifis known a~ the 'DI6'sequence (Venis and Napier, 1995) or 'Box A'7 and is thought to be involved in thebinding of the carboxylic acid group of IAA (Brown and Jones, 1994; Edgerton et al.,1994; Jones, 1994). Further evidence suggesting a link between ABPI and germinscome from circular dichroisn:t spectral analyses and structure predictive algorithms,neither of which support a significant arnount of helical secondary structure (Jones,1994). This is confirmed by the molecular modelling ofgermin reported by Ganeet aL(1998).

Phospholnallnose isomerases

Phospholnannose isomerases (PMIs) (EC 5.3.1.8) are enzylnes that catalyse theinterconversion of Inannose-6-phosphate and fructose-6-phosphate. Although theyare considered to be zinc-containing metalloproteins, a Fe(IIl)-hydroxyphenylalaninesite has been identified in the PMI from Candida 1I1bicalls \vhen expressed in E. coli(Proudfoot et aI., 1996; Smith et al. f 1997). On the basis of sequence comparison, thePMIs have been divided into three classes (Proudfoot et ill., 1994), within which theclass II enzymes are involved in a variety of pathways including capsular polysaccha­ride biosynthesis and D-mannose metabolism. They all include, toward theirC-terminus, two histidine-containing motifs similar in sequence and spacing to thosefound in the other proteins discussed in this review. One difference is that thehistidines of the first motif are often displaced to give a G(X)7HXH(X)9G sequence.

Notable amongst these enzymes, many of which are found in pathogenicallyimportant organisms, are the spore polysaccharide protein (P39631) from BacillussubtiUs, the Cap5F and Cap8F proteins from Staphylococcus aureus (Sau and Lee,1996) and the closely related (>90%) but possibly misnamed 'nucleotide binding'protein (S33518) from the mycoplasma Acholepillsma l(lidlawii~ Of this large groupofrelated proteins, the smallest ( 128 AA) is the Synechocystis PMI gene (gill 001180).Others include: a sequence (504426) from Synec:hococctlS; a sequence (created asSwissProt virtual sequence VIRT2200) in the upstream region of the aspartateracemase gene (D84067) from the archaeon Desulfllrococcus (Yohda et al., 1996)(see section on cryptic genes below); GDP-mannose pyrophosphorylase (Q46859)from Yersinia ellterocoliticCl; and P07874 - the 56KD bifunctional enzyme fromPseudolnonas aeruginea with both PMI and GDP..mannose pyrophosphorylase ac..tivities (May et al., 1994). This latter enzyme is involved in the polymerisation of theviscous mucoid exopolysaccharidet alginate~ a compound composed of 1,4-linkeda­L-guluronic acid and ~-D-mannuronicacid subunits. Its pathogenic significance lies inthe fact that it protects the bacterial cell from host immune responses and antibiotics,and thus is also the major cause ofmortality in patients suffering from cystic fibrosis.In contrast to their role in pathogenesis, bacterially produced alginates, along with

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Cupills 15

those from marine Inicroalgae (seaweeds), are of great economic importance in thefood indu~try (Renn, 1997).

There are also eukaryotic equivalents of these enzymes (e.g 553039 froln yeast) inwhich ei therone or both nlotifs are conserved. Related sequences from Caellor!1abditis(~legllIlS include t\VO with unknown function~ gi11707132, and gill 082118. It had beennoted previously (see attachment to the sequence submission) that the latter sequencecontained a domain with weak similarity to the acidic domain of a rice glutelinprecursor. In fact, it is rnore sinlilar to a region of the legumin precursor (S54207) fromMagnolia salicifo/ia and other species.

P()lykl~tide synth[ISeS

Anlongst the bacterial sequences with the closest homology to gerolin is cure(Q02586) froID Streptolllyces cyaneus (Bergh and Uhlen 1992a,b). The gene productis probably a cyclase (although its exact function is unknown) and is part of thesynthetic pathway of the antibiotic curamycin. This compound has a polyketideskeleton consisting of orsellinic acid - the simplest aromatic polyketide possible.Closely related genes with similar function from other StrejJtonzyces species includethose from S. c()elic()!or (X55942) (Davis and Chater, 1990), S. /lalsted;; (L05390)(Blanco el al., 1993), and S. glaucescens (M80674) (Bibb et aT., 1.989) and X77865(see Figure 4 in Blancoetal., 1993). This latter study also identified sequences closelyrelated to PGERISEHYHPYSE (residues 47-60 in L05390) (cf. motif 1) that areconserved amongst several ~-glucosidases from plants, fungi, and bacteria (e.g.P26208 from C/os/ridiul11 therl1zocelltll1'l).

Additional nlembcrs of this group of putative cyclases include the B. sub/ilissequence P54430 and the sequenceolif (gHI402862) (product Pepl) within U60777(Stnith ef al. 1993) from the cryptic transposon 'r04321, a part of the broad host rangeIncP beta plasmid R751 ofEnterobacter aerogenes. This Pep1 sequence (nucleotides.1704-2126) is situated between a transposase tnpA, and 0112 (product Pep2), aputative Inember of the LysR falnily of bacterial regulator proteins. The notesaccolnpanying the submission, whilst noting ·possible polyketide cyclase on basis ofweak similarity to TCI1ZJ of Streptol11yces glaucescells, M80674', did not recognizethat the closest protein relative to Pepl, as revealed by a BLASTP search. is anArabidopsis germin (U75187).

Another possible menlber of this class is the 143 AA protein encoded by sequencegil 1653078 from Synechocystis. The smallest (110-132 AA) proteins in this group aretwo from Erl'vinia cluy:ialltheI11i, first a 'pectin degradalion protein KDGF' (accessionQ05527) (Condemine and Robert-Baudouy, 1991) and secondly the sequencegil1772621 in L39897, one from Mycobacteriurll tuberculosis (gifI654019) (Philippet aI., 1996), and one from Bacillus subtiIis (giI1881251).

Epinzerases

Sequence analysis has revealed that the cupin protein superfamily include a series ofepiInerases involved in the synthesis ofbacterial exopolysaccharides. These enzymesinclude dTDP...4-dehydrorhamnose 3,5-epimerases such as the ExpA8 protein re­cently shown to be involved in the biosynthesis of galactoglucan (exopolysaccharide

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16 J.M. DUNWELL

II) in Rhizobiu11'1 nzelioti (Becker et lIl., 1997). The complete list of these enzymesincludes exwnples fronl E. coli (Stevenson et a/., 1994; Yao and Valvano, 1994;Marolda and Valvano, 1995), Xalltho11l01l0S clll1lpestris (Koplin et al., 1993) Lept­ospira illterrogcl1zs (Mitchison et al., 1997)9 Neisseria gOllorrhoeae (Robertson et al.,1994; Petering et al., 1996), N. rneningitidis (Hamlnerschmidt et al., 1994), Yersillia!Jseut!otubercu!os;s (Thorson et (ll., 1994), Sphi11g011JOIUlS (Yamazaki el a/., 1996)9Streptonryces griseus (Pissowotzki et al., 1991; Krugel el al... 1993), Sa/l1lollel/atJ'lJhilnuriutll (Jiang et al., 1991; Wang et (ll., 1992; Zhang et al., 1993), Shigella.f1exl11.?ri (Macphersonet al., 1994), Htlenzo/Jhilus llctil1ol1zycetel11C0l1litans (gil 1944162)and Synec!Ioc}'stis (gil 165678). The equivalent gene frOln C. elegal1~" is gill 049348.

A similarenzyme dTDP-6-deoxY..L-rnannose-dehydrogenase (giI1651977) is knownfrom Synechocystis.

Dioxygenases

Amongst these iron-containing enzymes is one class which contains the two histidine­containing nlotifs~ although the first motif has only a single histidine residue. Thisspecific class of enzylue cOIllprise the 3-hydroxyanthranilate 3,4-dioxygenases (3­HAO) that catalyse the synthesis of qUinolinic acid from 3-hydroxyanthranilic acid.Examples include a yeast sequence (accession P47096) and silnilar proteins from C.elegal"ls(Z70755) and rat (D44494) (Malherbeel11I., 1994). Additionally, the cysteinedioxygenases such as that from rat (P21816) (Hosokawa et (ll., 1990) clearly containan equivalent first ITIotif, though the second is either absent, or present as a weaklysimilar Inotif separated by a gap of approximately 40 residues.

Related proteins lvith no knO\VlZ ji-inctioll

Additional analysis identified a series ofprogressively smaller proteins, each of whichcontain motifs 1 and 2 (Dunwell and Gane, 1998). Included amongst these proteins,none of which has any known function, are two similar accessions (D90909, D9091 0)fronl Synec!locystis, the first of which has the Arabidopsis germin X91957 as theclosest relative~ and the second of which is similar to cytochrome c551 fromRhodo(.'oCCllS. Slightly shorter sequences include one from Myco!Jllcteriul11 tubercu­losis (gi12104394). two from the Inethanogenic archaeon MetluUZlJCOCcllsjallllllschii(Bult et (II., 1996), [gi11592216 (U67602) from predicled coding region MJ1618 andits close relative gil 1499583 (U67521 )], and two E. coli sequencest P38522 which issimilar to an aldehyde dehydrogenase (Heim and Strehler, 1991), and its longerversion - the immunity repressor protein (D90768).

The two sequences fronl M. jallnaschii have not yet been assigned a function,though it is to be expected that the type of metabolic reconstruction being devised bySelkov et al. (1997) will help towards that end.

The smallest (77 AA) of alll:hose identified is a membrane spanning protein (MSP)(Li and Strohl. 1996) (U37580) from Streptol12yces coelicolor. This protein, which hassimilarity to E. coli PM•• and also to an upstream sequence (SwissProt virtual sequenceVIRT17611) in the prismane gene (Z11975) froln Desulfovibrio desulfuricllllS(Stokkennans et ai., 1992) (see next section), is the putative progenitorof all these twomotif proteins, and subsequently of all the related cupin superfamily of proteins.

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CUIJins 17

Hypothetical clyplic genes

There are several exalnples of previously unknown, germin-like genes revealedrecently (J.M. Dunwell, unpublished results) by means ofTB'LASTN searches, whichare able to reveal putative coding regions in the upstream regions of other genes. Themost likely explanation for these cryptic ORFs, which may be found on either strandin any reading frarne, is the accidental ligation of DNA sequences during the cloningprocedures employed in the construction of eDNA libraries (Bhatia et al., )997).

Of the six exanlplcs described here, the most complete is that found in the upstreamsequence of the Arabidopsis polyubiquitin gene uhq4 (Burke et al., 1988). The 4024nucleotide cDNA sequence (giJ9875 18) from this study shows the coding region tostart at position 2649. Inspection of the ~upstream' sequence reveals that nucleotides1195 to 1873., with the insertion of a 'g' at position 1288 in order to overcome a frameshift error introduced by the sequencing procedure, encodes a germin-like proteinidentical to gHl755168 and that encoded by the Arabidopsis EST, ATfS 0248(gil 16847).

A very similar situation is present in the upstream sequence of a groESL operon(DI2677) froln 5)1llechocystis (Lehel et al., 1993). Nucleotides 3-208, with additionof an additional 'g' at position 104, encode a protein identical (with the exception ofa single Asp to Tyr mutation) to the C-tenninal69 amino acids encoded by gil1652486(accession D90905)1 a PMI (see above) similar to gillOOl J80 from the SaIne species.Perhaps coincidentally, it is reported that the correct folding of a recombinant type IPMI frolu Candida albicClI1S (Cleasby et al., 1996) in E: coli is increased by the co­expression of the GroES or GroEL chaperones (Proudfoot et aI., 1996).

The third example (VIRT28811) (Table 4) is encoded by nucleotides 338-1(negative strand) of the upstream sequence of gil560029 which contains at nucleotideposition 507-2363 a gene for a xylan 1,4-beta-xylosidase (Oh and Choi, 1994).Intriguingly, the closest neighbour of this cryptic sequence is the next sequencedescribed.

l~he fourth exalnple (VIRT2200) (Table 4) is found in the positive strand (nucle­otides 104-450) of the upstream sequence (although not in the same reaping frame) ofthe aspartate racemase gene from the archaeon Desu!furococcus strain SY (Yohdaetal., 1996). This sequence is most similar (50% similar over a 79 AA region) to thel:fbgene from Sahllonelill enlerica (see section on epimerases above).

The fifth exalnple (VIRT1761 I) (Table 4) is that from nucleotides 238-727 in theupstream sequence (same reading franle) of the prismane protein from Desulfovibriovulgaris (Stokkennans et al., 1992). Again, this derived sequence has another crypticsequence as its closest neighbour, in this instance a protein (VlRT20248) (Table 4) fromPseudonzoluls /elnoigllei encoded by the nucleotides 1618-1286 (negative strand) ofaccession U12977, between a glycerol-3-phosphate-dehydrogenase (giI531467) and apoly(3-hydroxybutyrate) depolymerase A (gi153 1466) (Jendrossek et at., 1995).

TWO-DOMAIN PROlEINS (BI-CUPINS)

Oxalate decarboxylase and related I1z;crobial proteins

The principal organisms responsible for the biodegradation of wood are white-rot andbrown-rot basidiomycete fungi (Dutton et al.1 1993; Dutton and Evans, 1996).

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Table 4. Putative cupin sequences encoded by cryptic genes in upstrea'll ·n()n-c()ding~ regions ufolher bacterial genes (J .M. Dunwell~ unpublished results)

>VIRT288I I, I 12 AA (Bacillus sleClrotherlllopllilus)FIFKI.Fl'TAFAGRLRRSGRIRRHSHPl.fEVNUl.AGSQVMIVGGKRYVOOroDllLVRPEDVHESRALGIEPMI'YYCLHFNVDEPVFASYCAVARTRSTPRTGRWPRSFGQA

>VIRT2200. 115 AA (f)e.\·uljur()cocclts)WGERVI<AElKNUDRGIYRKl..PLPEGa.F£GSYAQIVEVKfGQ1VKKl;YHLHQYElFYIMSGr~ICJEI'GYLARPGDIFLVKPKTVHWVINEKEEPFRLFVV KLNYKODDSV\V

>VIRT17611 163 AA (IJesu/fibrio desulfilrlclIlls)RPAAHQRKKHLPASSRPPERIl..RI.KSVTlVlDVHIPPWYlUIIKSKEGIMQRIJ",KNLDYATALPLAAQVrx:QPGQVAS~VQNDAVG1llFAFDKGEKl.c)AHfSIUDAFVLALEGQGQVI1I'lGQ1SPLKAGESnMPAGQPH..~"SA

VERFKMLLVVIFPPE

>VIRT20248 I] I AA (P.'teud(}mo/fas 1e.l1loigllei)MALPHASSGQLIDVRPLGSQLTSAPSRAlLKISGLELMHMW-PAGR1VPEHRVPGEC11QCIEGSVELTSHGNTQLMRAGDLVYFEGGVVHALHAQEDSSLLVTILMKHEA

Whereas the former type are able to degrade all components of the wood cell wall,brown-rot fungi remove the cellulosic fraction and leave lhe lignin undegraded. It hasbeen known for many years that most brown-rot fungi aCCUlnu)ate large quantities ofoxalic acid in certain culture media (Shimazono t 1955). In similar conditions, the\vhite rot organisms fail to show this acculDulation, because of the activity of oxalatedecarboxylase (ODC)-the enzyme (EC 4.1.1.2) that converts oxalic acid to fomu\teand carbon dioxide. (It has recently been shown by Micales (1995) that brown-rotfungi also produce an ODe enzyme.)

In cODlmercial terms, this enzyme has potential value (Hiatt and Owades, 1987) asan additive that can be supplied during the brewing process in order to reduce the levelof oxalate (to prevent 'gushing') (Haas and Fleischman, 1961) and to degrade'beerstone', crystals of calcium oxalate formed in the vat and therefore likely toobstruct pipe-work.

The Co/lybia velutipes ODe enzyme was first characterized in detail and cloned byMehta and Datta (1991) and its sequence recently published (Datta et al., 1996).Analysis of the protein sequence (Dunwell and Gane, 1998) has no\v revealed thesurprising fact that the protein is a duplicated one, with the two domains each showingsequence similarity to the ancestral germin protein (Baumlein et al., 1995). It istherefore quite closely related to the plant storage proteins such as vicilin and legumin,and is also related to the other oxalate degrading enzyme ox-ox (see above). Use of thegermin homology model (Gane et al., 1998) should enable predictions to be Inade ofthe structure of the active site ofthis ODe (Labrou and Clonis, 1995) in the near future.

Identification of this two-domain ODe in the fungus C. velutipes has also led to thediscovery of a closely related bacterial sequence, namely gil1652630 from theunicellular cyanobacterium Syllechocystis PCC6303 (Kaneko et al., 1996). l~his

hypothetical protein is encoded by nucleotides 15268-16452 of accession D90907and its closest neighbour as estimated by residues 250-381 is Arabidopsis germin-likeprotein U95046 (300/0 identical, 49% similar). Another example of duplication isprovided by a protein (accession P42106) (Yoshidaet al., 1995) of unknown functionfrom Bacillus subtilis.

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Cupins 19

Despite the fact that the critical residues wilhin each motif, and the spacing betweentnoti fs, have been conserved in all three examples~ there are a number of significantdifferences between composition of these proteins. First, the overlaps of the dupli­cated regions are of different length. Additionally, BLASTP searches with BEAUTYannotation (Worley et al., 1990) show that the stretches of sequence showingsignificant homology with other proteins are located primarily in the C-terrninalsection of oxalate decarboxylase, whereas they are located mostly in the N-terminalhalfof accession P421 06. The third protein, gil 1652630, has equal similarily (particu­larly to gennins/vicilins) in each half.

In terms of their evolution, there are a number of possible origins for this duplica­tion, which previously has never been found outside the plant kingdoln. The simplesthypothesis is that the two domains of each protein have diverged differentially after asingle duplication event Alternatively, duplication might have occurred three times,most recently in Syllechocystis (giI1652630) in \vhich the two domains show equalsimilarity to otherproteins. The third possibility is thal this latter protein is the only oneto be a product of duplication, and that the other two examples have instead evolvedfrom a homologous recombination event between two similar DNA sequences. In thiscase, gil 1652630 would be the direct and sole ancestor of the higher plant storageproteins (32/52 of its nearest neighbours are such proteins).

Sucrose binding protein (SBP)

Pfhe fact that sucrose-binding proteins (or at least the example from soybean, Q04672~GriIneset al., 1992) is a two-domain member ofthe germin family, was first reportedby Braun et af. (1996) in a study of a vicilin-like protein (Z50791) from the cycadZal1ziaftl1furac:ea. In a detailed anaJysis of sequencet these authors suggest that thereplacement ofa conserved proline residue (found in vicilins) with an isoleucine in theSBP C-terminal dOlnain Inight create stearic hindrances and therefore prevent thetrimerization of subunits typical of storage proteins.

Storage /Jroteins

Seed storage globulins are divided into two types, the viciIin-like 75, and the legumin­like lIS. The fonner 75 proteins are trimers assembled from two main types of 50 or70 kD subunits, whereas the subunits of lIS proteins are synthesized as a precur.sorwhich is processed into an acidic (l- and a basic J3-chain whilst remaining connectedby a disulphide bond. These subunits are then assembled to produce a hexamericholoprotein. Within the last few years it has been found that the genes coding for boththe legumin and viciIin subunits are the result of an internal duplication of a singledomain ancestral gene (Lawrence et aI., 1994). It was shown subsequently (Baumleinet al., 1995) that the spherulins and wheat gennins might also have evolved from thiscommon 'protogennin~ ancestor, for which various bacterial candidates have beenproposed by Dunwell and Gane (1998).

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Role of glycosylation

GERMIN AND GERMIN-LIKE PROTEINS

All exalnples ofgermin and its close relatives are glycosylated. It is notable, however,that the glycosylation site(s) vary in position within lhe protein, and perhaps could heused as a diagnostic feature to categorise the different classes of these proteins. Theon)y derailed study of the glycan side chains was that conducted on the wheat gennins(Jaikaran et al., 1990).

Several of the other single-domain gennin-like proteins, such as the ABPs, alsocontain glycosylation sites, with the position ofsuch sites being a feature ofeach groupof protein.

STORAGE PROTEINS

Phaseolin, the Inajor storage protein of bean, is encoded by a small gene falnilydivided into two highly homologous classes~ a and p (Slighlom et aI., 1985).Polypeptides of both classes are synthesized as two glycofornls bearing either one ortwo glycan side chains. This glycosylation is not required for the assembly of themonomers into the final trimeric structure (Vitaleet al., 1995) but itdoes affect the rateofassembly. More recently, it has been shown that glucose trimming of the side chainsis a key event in the asselnbly process (Lupatelli et a1., J997).

Structure predictions

As described above, germins possess sequence similarity with legumin and vicilinstorage proteins. Recently, this information was utilized to generate a 3D nlodel ofgermin (Gane et aL., 1998; see Figure J) from which a potential ox~ox active sitecould be predicted. This site, comprising a cluster of three histidine residues, waslocated within the conserved ~-barrel structure and was siinilar to those found incopper amine oxidases, although it should be noted that the metal cofactor of ox-oxhas never been unequivocally demonstrated. In additiont the nlodelling study used thepotential for charge-charge interactions bet\veen Dlonomers to produce a pentalnericasseIllbly, in which the ~-barrels remain parallel lo the five-fold axis, therebymaintaining solvent exposure for the predicted active sites.

This pentalneric structure of gerrnin is renliniscent of that found in the pentraxins,a family of vertebrate plasma proteins which include human C...reactive protein, theclassical cytokine-regulated acute phase protein produced in response to inflanuna­lion, and universally measured as an objective marker of disease activity (Emsley etal., 1994; Gewurz et al., 1995; Hohenester et at.., 1997; Srinavasan et llf., 1994). Likegermin, the pentraxins are protease resistant, and structural analysis reveals anothersimilarity in that their tertiary fold is very similar to that of another class of plantstorage protein, namely the legume lectins. Recently, several groups (Goodmanet al.,1996; Basile et al., 1997) have identified an additional class of ~long pentraxins'.Although only the C-tenninus shows features of the pentraxin family, they are stillthought to form pentameric complexes. In some ways these extended proteins areanalogous to the two...domain, bi-cupin proteins.

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Cupins 21

Figure 1. Proposed molecular model of the gcrrnin molecule. based on the honlology with the ~loragc

protein vicilin (Gane fH a/.• 1998). The arrow indicates the position of the histidine cluster, the suggestedactive site of the oxalate oxidase (courtesy Jim \Varwickcr).

It is expected that the 3D tllodel of gennin will soon be used to generate structuralpredictions of other proteins in the. superfalllily.

Concluding remarks

'The cereal gernlin proteins, along \vith their close relatives from gymnosperms anddicotyledonous plants, have ernerged recently fronl a position of being considered asinteresting proteins with no kno\vn function, to being identified as enzyJnes concerned\vith cell \vall structure (Lane, 1994), fungal defence (Dumaset al., 1993; Zhanget al.,1995~ HurkJnan and Tanaka, 1996b), salt tolerance (Michalowski and Bohnert, 1992;Hurknuln and Tanaka, 1996a), floral induction (Heintzen el al., 1994~ Dna et al.,1996), and as l11arkers of somatic embryogenesis (Donlon et al., 1995). In addition,knowledge of their ancestral relationship to plant storage proteins (DunweJl and Ganc,1998) has been used to generate a 3D model (Ganeet al., 1998) and identify details ofthe potential active site. Together with the identification of several other relatedproteins in different enzylue classes - a finding predicted by Lane (1994) and silnilarto the results of Murzin (1993) and Babbit et al. (1995) - these new observations willundoubtedly lead to a further rapid expansion in fundalnental and applied interest inthis Illost fascinating group of proteins.

Ackno\\'ledgements

I would like to thank the Biotechnology and Biological Sciences Research Council,

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and ZENECA pic for funding. 1 am also most grateful to Dr Jinl Warwicker forprovision of Figure /.

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