mechanisms and pathways from recent deoxysugar biosynthesis research

642

Mechanisms and pathways from recent deoxysugar biosynthesis research David A Johnson and Hung-wen In the past few years, there have been many important advances in our understanding of the biosynthesis of deoxysugars. Mechanistic studies have shed light on how enzymes can cleave C - O bonds, epimerize the configuration of substituents and reduce keto groups to make deoxysugars. Exciting progress has also been made in our comprehension of the genetics of deoxysugar biosynthesis in antibiotics. All this information is important for potential medical and biotechnological applications, such as drug discovery based on combinatorial biology.

Liu*

Addresses Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA *e-mail: [email protected]

Current Opinion in Chemical Biology 1998, 2:642-649

http://biomednet.com/elecref/1367593100200642

© Current Biology Ltd ISSN 1367-5931

Abbreviations CDP cytidine diphosphate FAD flavin adenine dinucleotide PMP pyridoxamine 5'-phosphate U DP uridine diphosphate

I n t r o d u c t i o n The deoxysugars, long recognized as an important class of carbohydrates, have enjoyed increasingly widespread appreciation as essential biological molecules playing a great number of roles in many physiologically significant reactions, including celhflar adhesion, cell-cell interac- tions, immune response, fertilization and target recognition of toxins, antibiotics and microorganisms [1,2°°]. A wide variety of deoxysugars are found in lipopolysaccharides, glycoproteins, glycolipids and many secondary metabolites. In many cases, these unusual sugars have been shown to be indispensable for the activity of the parent molecule [2°°]. Because modifying the struc- ture and/or composition of the deoxysugar components holds promise for varying and/or enhancing the biological activities of the parent systems, it is pivotal to have a complete understanding of the biosynthesis of these unusual sugars so that suitable strategies to control, mimic or alter their formation can be developed. Efforts directed toward these goals have achieved some notable results. For example, sequencing and analysis of various antibiotic biosynthetic gene clusters and the correlation of blocked mutants with phenotypes have provided critical evidence allowing the identification of a number of complete sugar biosynthetic gene clusters. Detailed mechanistic information has also become available for a few enzymes involved in the formation of deoxysugars. Some of the more inter- esting examples reported in the past two to three years are

reviewed here. Much of the early work on deoxysugar biosynthesis has been summarized in a recent review by Kirschning et al. [3°°].

M e c h a n i s m s o f d e o x y s u g a r b i o s y n t h e t i c e n z y m e s Although our knowledge of the pathways of deoxysugar formation and the mechanisms of most transformations involved in deoxysugar biosynthesis remains limited, significant progress has been made in the past decade in our understanding of a few specific enzymes catalyzing the key conversions yielding 2-deoxyribose, 6-deoxyhexoses and 3,6-dideoxyhexoses. Summarized below are the latest results concerning the modes of action of the C6 and C3 deoxygenations leading to 6-deoxyhexoses and 3,6- dideoxyhexoses. Brief descriptions of the mechanisms of cytidine diphosphate (CDP)-paratose synthase and uridine diphosphate (UDP)-N-acetylglucosamine 2-epimerase are also included. The study of C2 deoxygenation, catalyzed by ribonucleotide reductase, is the subject of Lawrence and Stubbe's review in this issue (pp 650-655), and will not be covered here.

CDP-D-glucose 4,6-dehydratase As depicted in Figure 1, the biosynthesis of 6-deoxyhexose begins with the conversion of hexose-l-phosphate (e.g. C~-D- glucose-l-phosphate, 1) to a nucleotidyl diphosphohexose (e.g. CDP-c~-D-glucose, 2) by a nucleotidyl transferase (Ep). This compound then undergoes C5/C6 dehydration, catalyzed by a NAD+-dependent nucleotidyl diphosphohexose 4,6-dehydratase (Eod), to form a 4-keto-6-deoxyhexose (3).

Most recent studies on the biosynthesis of 6-deoxysugars have focused on elucidating the mechanism of CDP-D- glucose 4,6-dehydratase, specifically the Eod of Yersinia pseudotuberculosis that is involved in the biosynthesis of CDP-L-ascarylose (8, Figure 1). Early studies of this homodimeric enzyme had confirmed that the C6 deoxygenation proceeds via three discrete steps: oxidation of CDP-D-glucose to a 4-keto-glucose, C5/C6 dehydration to a 4-keto-AS,6-glucoseen intermediate and reduction at C6 to give the final product 3 [4]. This intramolecular oxidation/reduction is accomplished by an internal hydrogen transfer from C4 of the substrate 2 to C6 of the resulting product 3, and the hydride carrier is an enzyme-bound NAD+; however, CDP-D-glucose 4,6-dehydratase binds only one equivalent of NAD + per mole of enzyme dimer and, unlike other enzymes of the same class, displays a unique NAD + requirement for fifll catalytic activity [4].

Recent analysis of the cofactor- and substrate-binding characteristics of CDP-D-glucose 4,6-dehydratase has defined the presence of two binding sites for both

Deoxysugar biosynthesis Johnson and Liu 643

Figure 1

OHO0 CTP

HO OP03 1

#- Me OH 11

OCDP

JOH Eod E 1 H 0 . ~ . 0 ~ O_ Me

H O ~ NAD+ H PMP H°dcDp HOocDp 2 3

Me 10

HO OCDP

Me O- H, + Me

H-N+ Z

_O~PO ~ 4 HoOCDP - 3

E3 I NADH

o~Me n H20 Me O- H + Me

TdC CDP = 0 3 PO

H O ~ 9 I M e ~ O C D P Ered

O ~ NAD(P)H HO (DCDP 7 OH

. e __ OODP OH 8

Current Opinion in Chemical Biology

General pathways for 3,6-dideoxysugar biosynthesis. c~-D-glucose-1 -phosphate 1 is converted to CDP-D-glucose 2, by a nucleotidyl transferase (Ep). An NAD+-dependent nucleotidyl diphosphohexose 4,6-dehydratase (Eod) catalyzes the dehydration of 2 to 4-keto-6-deoxyhexose, 3, which is then converted to 3,6-dideoxyhexose 6 by the sequential activities of a dehydrase (E 1 ) and a reductase (E 3) via a A&4-glucoseen intermediate 4. After generation of the common

intermediate 6, by hydrolysis of the PMP-product Schiff base 5 in the E 1 active site, subsequent epimerization (e.g. 6 to 7 catalyzed by epimerase E e ) and/or stereospecific reduction (e g 7 to 8 catalyzed by reductase p • .

Ered) result in the various isomers of 3,6-dideoxyhexoses, ascarylose (8), abequose (9), paratose (1 O) and tyvelose (11). Note that the pathway for another 3,6-dideoxyhexose, colitose, is initiated with CDP-D-mannose and is omitted from this figure. Me, methyl.

CDP-D-glucose and NAD + per enzyme molecule [5]. The large anticooperativity found for NAD + binding (dissociation constants K 1 = 40 nM and K z = 540 nM) may explain why the cofactor-binding sites of wild-type Eod are only half occupied. Further examination also revealed that the purified Eod contains sequestered NADH and that the affinity of Eod for NADH (K 1 = 0.21 nM, K z = 7.46 nM) is much higher than that for NAD +. Thus, it is possible that Eod'S half-site saturation of NAD + per enzyme dimer may also be attributed to a significant portion of the cofactor- binding sites being occupied by NADH.

Cytidine diphosphate-6-deoxy-L-threo-D-glycero-4- hexulose-3-dehydrase and its reductase The step immediately following the Eod reaction in the biosynthesis of 3,6-dideoxyhexoses is C3 deoxygenation. This reaction is catalyzed by the concerted activities of CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase (El), a pyridoxamine S'-phosphate (PMP)-dependent Fe-S protein, and E 1 reductase (E3), an Fe-S flavoprotein [6]. As shown in Figure 2, the catalysis is initiated by the Schiff base formation (12) between 6-deoxy-4-ketohexose 3 and PMP in the active site of E v Subsequent abstraction of the C4' pro-S hydrogen atom from 12 by His220 in the active site triggers expulsion of the 3OH group to give a A3,4-glu - coseen intermediate 4 [7]. The reaction is driven to completion when 4 is reduced to 5 by E 3. Because the [2Fe-2S] centers in both these enzymes are capable of only

one-electron chemistry, the reduction of the A3,4-glucoseen intermediate 4 must involve stepwise electron transfers.

To better understand the electron transfers carried out by these two enzymes, the potentials of the E 1 and E 3 redox cofactors were recently determined [8]. The data indicated that the redox properties of this system may be regulated by pH and the electron transfer between the E 3 redox centers may be prototropically controlled. In a separate study of the reductive half-reaction of E 3, the hydride transfer from NADH to flavin adenine dinucleotide (FAD) was established to proceed with a tenfold kinetic isotope effect when (4R)-[ZH]NADH is substituted for NADH [9]. Interestingly, the extent of the intramolecular electron transfer from the reduced FAD to the [2Fe-2S] center was found to be pH- dependent with a pK a of 7.3, which may represent the ionization state of the N1 position of the FAD hydroquinone of E 3. Evidence supporting E1-E 3 complex formation as a prerequisite for the C3 deoxygenation activity was also reported [10]. The apparent dissociation constant of the E1-E 3 complex was estimated to be 288 nM, and the stoi- chiometry between E 3 and E 1 of this complex was deduced as 1.7:1. By using a two-hybrid system that screens for inter- actions between two proteins coexpressed in yeast, the E 1-E3 complex formation in vivo was also firmly established.

Passage of two electrons from N A D H through these iron centers to reduce the dehydration product 4 (Figure 1)

644 Mechanisms

Figure 2

0 % _ ~ 0 PMP

3

E3 NAD + FADhqy [2Fe-2S12~-,~

NADH J ~ FAD°x-J~'le/le [2Fe-2S]1+ j

O_ Me _ PMP

" ~ H20

HO £)CDP 6

Me O- H+ Me .0 Me O-H+ Me 0 51

= 0 3 ~ k H 1 2 _01~ 4 ~.~/3 - I

Me 0 H+ Me 0 H " ' ~ k ; N ~ " ~ O C D P ~ " HO H Me N ~ 30~ H'I~ ~ ' ) ~ , ~ . , , . ~ . , Me 0

=03P0.5 1 3 ~ H + HO VvaaP =03FO 4

f [2Fe_2S]1+ ..N f Me 0 H I ~ . ~ / / ~

M_ [2Fe_2S]2+...) ., H Nx /X~J" "~ ILlO OCDP

1 ~ = O 3 FO~ 1 4


Proposed detailed catalytic mechanism of the coupled E 1-E 3 reaction in 3,6-dideoxysugar biosynthesis. In the course of C3 deoxygenation of 6-deoxy-4-ketohexose 3, the E 1 reductase E 3 transfers reducing equivalents from NADH in a stepwise manner through the flavin adenine dinucleotide (FAD) and [2Fe-2S] centers in E 3 and El, to the substrate. After formation of the PMP- substrate complex 12 and dehydration of C3 to form the PMP-Aa,4-glucoseen intermediate 4, presumably there is a tautomerization to an electrophilic quinone

methide 13 before electron transfer to the substrate. The obligatory single electron transfer results in the transient formation of a PMP-A&4-glucoseen radical intermediate (14). Once radical 14 is reduced to a quinone anion by a second electron, protonation at C4' of PMP leads to the PMP- product complex 5, which is subsequently hydrolyzed to form 6 amd regenerate the PMP cofactor, e, electron; El, CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase; hq, hydroquinone; Me, methyl; ox, oxidised.

must involve radical intermediates because these Fe-S centers are one-electron carriers. Previous studies of the reduction of E 1 with dithionite in the presence of substrate 3 had afforded preliminary evidence for the formation of a radical intermediate [11]. Using N A D H as the reductant in the coupled El-E3 reaction, the kinetics of this radical intermediate were fi~rther analyzed using both stopped-flow spectrophotometry and rapid freeze- quench electron paramagnetic resonance spectroscopy (EPR) under aerobic, as well as hypoxic, conditions [12]. A sharp signal a tg = 2.003, which is typical for organic radicals, appeared with a maximum intensity at -150 ms. Stopped-f low UV-visible analysis of the reaction revealed that one of the optically detectable intermediates has a time course nearly identical to that of the radical detected by rapid freeze-quench EPR. T h e UV-visible spectrum of this intermediate displays a maximum at 456 nm, with a shoulder at 425 nm. Such characteristics support a phenoxyl radical 14, whose formation has been hypothesized to involve a tautomerization of the PMP-A3,4-glucoseen intermediate to a PMP-quinone methide species 13 (Figure 2), which

then serves as the electron acceptor during the reduction. Incubation of E1-E s with a new coenzyme B 6 analog, 3- deoxy-3-fluoro-PMP (3-F-PMP), which cannot undergo such a tautomerization, was at tempted in order to gain support for the proposed mechanism [13]; however, because of the low pK a (-2.91) of the ring nitrogen atom of 3-F-PMR this new coenzyme B 6 analog is not an effec- tive electron sink and fails to bind E 1 properly in the active site. Nevertheless, the currently available data are consistent with an electron transfer pathway that includes a radical intermediate with the unpaired spin localized on the substrate-cofactor complex 14 (Figure 2). These studies add the PMP-A3,4-glucoseen radical to the growing list of mechanistically important bio-organic radical intermediates that have recently been discovered (J Stubbe, W van der Donk; see Note added in proof).

Study of CDP-o-paratose synthase and UDP-N- acetylglucosamine 2-epimerase As illustrated in Figure 1, reduction of the 4-keto group of the E]-E 3 product 6, with or without prior epimerization at C3 and/or C5, results in the formation of various isomers of


F igure 3

UDP-GIcNAc Me H 2-epimerase

- _ ~ . . _ . 0 0 *H20

HO

AcHN I II II O - - P - O - - P - - O - - U (.' ' 15 O-- O--

bridging position

Me NHAc nonbridging position

O O *H I II II

0 P 0 P 0 U I I

1 6 O - O -

Enz-B1

Me H

+ AcHN Enzq3T*H

17

+ Enz-B~- H

Me

AcHN + Enz-Bu*H 1 8

0 0 II II

'-0-- P - O-- P-- O- -U I I

O-- O--

O O II II

'--0-- P - O-- P-- O- -U I I

O-- O--

Postulated reaction mechanism of U D P-N-acetylglucosamine 2-epimerase, which catalyzes the interconversion of U DP-N- acetylglucosamine (15, U DP-GIcNAc) and U DP-N-acetylmannosamine (16). Species within the brackets are enzyme-bound. After formation of the oxocarbenium intermediate 17, an active-site base (B 1) abstracts

the C2 proton to form a 2-acetamidoglucal intermediate 18, which is stereospecifically protonated by an active-site base (B 2) to yield 16. It has not been determined whether B 1 and B 2 are the same residue. Adapted from [17"] and reproduced with permission. Ac, acetyl; Enz, enzyme; GIc, glucosamine; U, uracil.

3,6-dideoxyhexoses- ascarylose (8), abequose (9), paratose (10) and tyvelose (11). Although little is known about the catalytic properties of the corresponding epimerases and reductases [14,15], the reductase encoded by the rfbS gene of Salmonella ~.phi in the paratose pathway, known as CDP-D- paratose synthase, has been overexpressed, purified and studied in detail [16°]. This homodimeric enzyme, catalyzing a hydride transfer of the pro-S hydrogen from NADH to C4 of the substrate 6, utilizes a Theorell-Chance mechanism. This study represents the first detailed characterization of this type of ketohexose reductase, and those from other deoxysugar biosynthetic pathways may share similar properties with CDP-D-paratose synthase.

With regard to the epimerization step in the deoxysugar biosynthesis pathways, the mechanism of UDP-N-acetylglucosamine 2-epimerase, which catalyzes the interconversion of UDP-N-acetylglucosamine (15, Figure 3) and UDP-N-acetylmannosamine (16, Figure 3), has been elucidated [17°]. The incorporation of a solvent hydrogen at C2 of both epimers, and the scrambling of the 180 label from the sugar-UDP bridging position into the nonbridging diphosphate positions during the epimerization lent cre- dence to the mechanism shown in Figure 3. The proposed route involves temporary cleavage of the anomeric C-O bond, perhaps initiated by a cationic elimination via an oxocarbenium species (17), to generate 2-acetamidoglucal (18) and UDP as enzyme-bound intermediates. Although this reaction and the C2 epimerization converting CDP-paratose 10 to CDP-tyvelose 11 (Figure 1) are chemically alike, the translated sequence of the enzyme (RfbE [18]) catalyzing the conversion to CDP-tyvelose contains a Rossmann fold

(a possible NAD+-binding site [19]) that is lacking in UDP- N-acetylglucosamine 2-epimerase. Hence, the mechanism of CDP-D-tyvelose synthase may be distinct from that of UDP-N-acetylglucosamine 2-epimerase.

Genet ics of d e o x y s u g a r b iosynthes is Our knowledge of the genetics of deoxysugar biosynthesis has grown considerably in the past few years. The recent major advances include the identification of some genes needed for the production of deoxysugar components in avilamycin of Streptomyces viridochromogenes Tii57 [20], mithramycin of Streptomyces argillaceus [21], nogalamycin of Streptomyces nogalater [22], daunorubicin of Streptomyces peucetius [23] and a few lipopolysaccharides of Azotobacter vindandii, Leptospira interrogans and Streptococcus mutans [24-27]. Such rapid progress is quite remarkable considering that the sugar biosynthetic genes in these clusters are usually scattered and, hence, are dif- ficult to distinguish from those encoding regulatol T or aglycone modification enzymes that are also interspersed in the same region. A notable case is the identification of a set of genes (the cluster strO-stsABCDEFG) that encode proteins for streptomycin production in Streptomyces griseus [28°]. Sequence analysis revealed that StsA and StsC proteins are members of a new class of aminotransferases that are used mainly in carbohydrate biosynthetic pathways. Purification of the StsC protein permitted unambiguous assignment of StsC as the L-glutamine: scyllo-inosose aminotransferase, which catalyzes the first cyclitol transamination reaction in the biosynthesis of the strepti- dine subunit of streptomycin [28°]. Because genes related to stsA and stsC also occur in actinomycete producers of

646 Mechanisms

Figure 4

OH

H HO OP03 = 0

M e ~ Q q ,, Me

Me . . . . . . . . IV Et',~ L ' -2Me~. ',,,,,

O" ~Me ,,, 0

EryB~" ]

OH

OH - - MOo-

HO OTDP

OH [HO 211

....... ° I ] 0 OH 201

OH N Me--7-- ] O~/--.OTDP EryBIV M e ~ O T D P

Me (EryBII) 0 Me

"

HO ~ , , ' ~ OP03 = %40T P

0 Me 0 EryCI__.....~I Me EryCIV

H O ~ H O 0 htO' ..-k--...~.jo~O OT EryCV

OTDP D P % Me

Me Me OTDP . , , C l i i ,,cvI j

- N(3 I - HO ] EryCI " ' - OTDP OTDP

0 Me 0 EryBI M e ~ O T D P EryBVll H O ~ _ _ - - ~

OHO OTDP

EryBII ) 0 Me 0 EryBVl O~---.~e/o J (EryBIV)

HO-~ 7H2----~ HO Q.~__ ~ HO OTDP OTDP


Proposed biosynthetic routes to mycarose (20) and desosamine (21) in erythromycin (19) produced by Saccharopolyspora erythraea, as delineated by Summers et aL [30"'], with genetic loci (EryCII, EryCIV, EryCV, and so on) assigned to each step. The assignments of EryBII and EryBIV (shown in brackets) are uncertain and may be reversed.

The routes proposed by Gaisser et al. [29"'] are not shown. Their proposal is similar, except for differences in the order of some steps in the biosynthetic sequence. For a detailed description of these pathways, see reference sources. Me, methyl; TDP, 2'-deoxythymidine diphosphate.

other diaminocyclitol aminoglycosides such as neomycins, kanamycins and hygromycin B, the StsC and StsA proteins may be considered as representatives of aminoglycoside-specific aminotransferases.

Another significant example is the sequencing and analysis of genes from the erythromycin (19) biosynthetic gene cluster of Saccharopolyspora erythraea that are involved in L-mycarose (20) and D-desosamine (21) production (Figure 4) [29"',30"]. This work was accomplished independently by two research teams, and the pathways proposed by each team for the formation of these sugars, as shown in Figure 4, are essentially identical. Part of the scheme is still ambiguous, however, because the fimction of several genes and portions of the reaction sequence cannot yet be clearly defined. In another report [31°], all the genes involved in the biosynthesis of L-mycaminose (22) from tylosin (23) were located and analyzed using sequence comparisons and genetic disruption experiments. The proposed pathway for mycaminose formation is depicted in Figure 5.

Early studies of the genetics of the daunorubicin-producing strain, S. peucetius, had led to the identification of

several genes within the dnm (formerly dnr) gene cluster that are involved in the biosynthesis of daunosamine, the 2,3,6-trideoxy-3-aminohexose component of daunorubicin [32-36]. Interestingly, a recent report on the characterization of the dnmZ gene in this cluster showed that though DnmZ is essential for the synthesis or attachment of daunosamine to the aglycone, e-hodomycinone, this protein shares significant similarity with acyl-CoA dehydrogenases [23]. Thus, the role of this putative flavoprotein in daunosamine biosynthesis is not obvious, and its identity must await the biochemical characterization of the purified DnmZ protein in the filture.

Conclusions It is evident that our knowledge of the biosynthesis of deoxysugars, especially their genetics and pathways, has accumulated rapidly over the past few years [3"',37]. A number of important deoxysugar biosynthetic genes have been cloned and identified, and mechanistic studies of the expressed enzymes have provided fresh insights into deoxysugar biosynthesis. T h e examples dis- cussed above highlight both the growing interest and progress in this critical field. Future efforts will be aimed at confirming the genetic assignments, determining the


Figure 5

) ) ~ TylA2 O- Me

D Ho H° °oTDP 40

- . - 1 TylM2 Me TylM1 ,o e2 i " " • , - - H O - - X < / O - -

2 2 [v'e } I IQH HOU/-' OTDP

Me 2 0

Me

O" HO (STDP

I TylB

Me

2 HO ()TDP

Proposed biosynthetic route to mycaminose (22) in tylosin (23) produced by Streptomyces fradiae, with genetic loci (TylA1, TylA2, and so on) assigned to each step. Gandecha et al. [31 °] have determined that TylM1 and TylM2 serve as the methyltransferase and

a glycosyltransferase, respectively, in this pathway. Me, methyl; TDP, 2'-deoxythymidine diphosphate. Adapted from [31 "] and reproduced with permission.

enzyme-subst ra te relationships, and establishing the actual sequence of the individual steps by directly exam- ining the activities of the gene products.

Considering that the deoxysugar components of most pharmacologically usefifl natural products are indispensable to their activities [2"], experiments have been at tempted to shuffle the sugar biosynthetic genes from different pathways to produce new compounds that differ in their sugar content or the aglycone core [38,39]. Such an approach has met with initial success in generating novel hybrid glycopeptide antibiotics by using different glycosyltransferases from Amycolatopsis oriental# strains that produce vancomycin or a related glucopeptide, A82846 [40"]. Another example involves the direct modification of the deoxysugar attached to doxorubicin and daunorubicin [41"'], two popular anthracycline antibiotics used in cancer treatments, by genetic engineering techniques. These experiments were the first to demonstrate the potential of combinatorial biosynthesis using bacterial sugar biosynthetic genes. Future experiments in this field designed on the basis of established deoxysugar biosynthetic pathways, along with the various emerging antibiotic biosynthetic routes, offer a tremendous potential to develop an array of metabolites that possess new and exciting biological activities.

Note added in proof The paper referred to as (J Stubbe, W van der Donk; see Note added in proof) has now been published [42].

Acknowledgements This work was supported by grants from the National Institutes of Heal th (GM 35906 and 54346). DA Johnson was a recipient of a National Institutes of Heal th biotechnology training grant (2 T32 GM08347).

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:

• of special interest • • of outstanding interest

1. Varki A: Biological roles of oligosaccharides: all of the theories are correct. Glycobiology 1993, 3:97-130.

2. Weymouth-Wilson AC: The role of carbohydrates in biologically °° active natural products. Nat Prod Rep 1997, 14:99-110. Comprehensive review of recent literature describing work aimed at determining the function of sugars in natural products, mostly antibiotics.

3. Kirschning A, Bechthold AF-W, Rohr J: Chemical and biochemical °° aspects of deoxysugars and deoxysugar oligosaccharides.

Top Curr Chem 1997, 188:1-84. This is an exhaustive review of literature up to early 1996 pertaining to the mechanisms and genetics of deoxysugar biosynthesis, as well as organic and chemoenzymatic synthetic techniques.

4. Yu Y, Russell RN, Thorson JS, Liu L-d, Liu H-w: Mechanistic studies of the biosynthesis of 3,6-dideoxyhexoses in Yersinia pseudotuberculosis. J Biol Chem 1992, 267:5868-5875.

5. He X, Thorson JS, Liu H-w: Probing the coenzyme and substrate binding events of CDP-D-glucose 4,6-dehydratase: mechanistic implications. Biochemistry 1996, 35:4721-4731.

6. Liu H-w, Thorson JS, Miller VP, Kelley TM, Lei Y, Ploux O, He X, Yang D-y: Mechanistic studies of the biosynthesis of 3,6-dideoxysugars in bacteria: exploration of a novel C - O bond cleavage event. J Chin Chem Soc 1995, 42:627-636.

7. Lei Y, Ploux O, Liu H-w: Mechanistic studies on CDP-6-deoxy-L- threo-D-glycero-4-hexulose 3-dehydrase: identification of His-220 as the active-site base by chemical modification and site-directed mutagenesis. Biochemistry 1995, 34:4643-4654.

8. Burns KD, Pieper PA, Liu H-w, Stankovich MT: Studies of the redox properties of CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3- dehydrase (E 1) and CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3- dehydrase reductase (E3): two important enzymes involved in the biosynthesis of ascarylose. Biochemistry 1 996, 35:7879-7889.

9. Gassner GT, Johnson DA, Liu H-w, Ballou DP: Kinetics of the reductive half-reaction of the iron-sulfur flavoenzyme CDP-6- deoxy-L-threo-D-glycero-4-hexulose- 3-dehydrase reductase. Biochemistry 1996, 35:7752-7761.

10. Chen XMH, Ploux O, Liu H-w: Biosynthesis of 3,6-dideoxyhexoses: in vivo and in vitro evidence for protein-protein interaction

648 Mechan isms

between CDP-6-deoxy-L-threo-D-glycero-4-hexulose 3-dehydrase (E 1) and its reductase (E3), Biochemistry 1996, 35:16412-16420,

11, Thorson JS, Liu H-w: Coenzyme B s as a redox cofactor: a new role for an old coenzyme? J Am Chem Sac 1993, 115:12177-12178,

12. Johnson DA, Gassner GT, Bandarian V, Ruzicka FJ, Ballou DP, Reed GH, Liu H-w: Kinetic characterization of an organic radical in the ascarylose biosynthetic pathway. Biochemistry 1996, 35:15846-15856.

13. Pieper PA, Yang D-y, Zhou H-q, Liu H-w: 3-Deoxy-3- f luoropyr idoxamine 5'-phosphate: synthesis and chemical and biological propert ies of a coenzyme B s analog. JAm Chem Sac 1997, 119:1809-1817.

14. Thorson JS, Lo SF, Liu H-w: Molecular basis of 3,6-dideoxyhexose biosynthesis: elucidation of CDP-ascarylose biosynthetic genes and their relationship to other 3,6-dideoxyhexose pathways. J Am Chem Sac 1993, 115:5827-5829.

15. Thorson JS, Lo SF, Ploux O, He X, Liu H-w: Studies of the biosynthesis of 3,6-dideoxyhexoses: molecular cloning and characterization of the asc (ascarylose) region from Yersinia pseudotuberculosis serogroup VA. J Bacterial 1994, 176:5483-5493.

16. Hallis TM, Lei Y, Que NLS, Liu H-w: Mechanistic studies of the • biosynthesis of paratose: purif ication and characterization of

CDP-paratose synthase. Biochemistry 1998, 37:4935-4945. The characterization of the first pure ketohexose reductase is described in this paper. Cytidine diphosphate paratose synthase is a new member of the short-chain dehydrogenase family and it utilizes a TheorelI-Chance mechanism. This is likely to be the prototype enzyme for ketohexose reductases in deoxysugar biosynthesis.

17. Morgan PM, Sala RF, Tanner ME: Eliminations in the reactions • catalyzed by UDP-N-acetylglucosamine 2-epimerase. J Am Chem

Sac 1997, 119:10269-10277. This report on the mechanism of an epimerase in an aminosugar biosynthetic pathway is important because the epimerization occurs at a stereocenter with an unactivated proton. The intriguing mechanism proceeds via cleavage of the anomeric C - O bond, with 2-acetamidoglucal and uridine diphosphate as enzyme-bound intermediates. It is suggested that cationic eliminations via oxocarbenium intermediates are involved in the reaction.

18. Verma N, Reeves P: Identification and sequence of rfbS and rfbE, which determine antigenic specificity of group A and group D Salmonellae. J Bacterial 1989, 171:5694-5701.

19. Rossmann MG, Liljas A, BrAnd6n C-I, Banaszak L J: Evolutionary and structural relationships among dehydrogenases. In The Enzymes, 3rd edn. Edited by Boyer PD. New York: Academic Press; 1975:61-102.

20. Gaisser S, Trefzer A, Stockert S, Kirschning A, Bechthold A: Cloning of an avilamycin biosynthetic gene cluster from Streptomyces viridochromogenes T/i57, J Bacteria/1997, 179:6271-6278,

21. Lombo F, Siems K, Bra~a AF, M6ndez C, Bindseil K, Salas JA: Cloning and insertional inactivation of Streptomyces argillaceus genes involved in the earliest steps of biosynthesis of the sugar moieties of the anti tumor polyketide mithramycin, J Bacteria/ 1997, 179:3354-3357.

22. Torkkell S, Ylihonko K, Hakala J, Skurnik M, M~nts~l~ P: Characterization of Streptornyces nogalater genes encoding enzymes involved in glycosylation steps in nogalamycin biosynthesis. Mol Gen Genet 1997, 256:203-209.

23. Otten S, Gallo MA, Madduri K, Liu X, Hutchinson CR: Cloning and characterization of the Streptornyces peutetius dnrnZUV genes encoding three enzymes required for biosynthesis of the daunorubicin precursor thymidine diphospho-L-daunosamine. J Bacterial 1997, 179:4446-4450.

24. Gavini N, Hausman BS, Pulakat L, Schreiner RP, Williamson JA: Identification and mutational analysis of rfbG, the gene encoding CDP-D-glucose-4,6-dehydratase, isolated from free living soil bacterium Azotobacter vine/and#. Biachem Biaphys Res Cammun 1997, 240:153-161.

25. Mitchison M, Bulach DM, Vinh T, Rajakumar K, Faine S, Adler B: Identification and characterization of the dTDP-rhamnose biosynthesis and transfer genes of the lipopolysaccharide-related rfb locus in Leptospira interrogans serovar Copenhageni. J Bacterial 1997, 179:1262-1267.

26. Tsukioka Y, Yamashita Y, Oho T, Nakano Y, Koga T: Biological function of the dTDP-rhamnose synthesis pathway in Streptococcus rnutans. J Bacteriol 1997, 179:1126-1134.

27. Tsukioka Y, Yamashita Y, Nakano Y, Oho T, Koga T: Identification of a fourth gene involved in dTDP-rhamnose synthesis in Streptococcus mutans. J Bacterial 1997, 179:4411-4414.

28. Ahlert J, Distler J, Mansouri K, Piepersberg W: Identification of stsC, • the gene encoding the L-glutamine: scyllo-inosose

aminotransferase from streptomycin-producing Streptomycetes. Arch Micrabial 1 997, 168:102-113.

Reported herein are the identification of eight new genes, strO-stsABCDEFG, in the cluster that encodes proteins for streptomycin production in Streptamyces griseus. Two proteins, StsA and StsC, are members of a new class of pyridoxal 5'-phosphate-dependent aminotransferases. StsC was purified, and its aminotransferase activity was demonstrated. This is the first report ascertaining the aminotransferase activity of this class of enzyme.

29. Gaisser S, B6hm GA, Cort6s J, Leadlay PF: Analysis of seven genes • • from the eryAI-eryK region of the erythromycin biosynthetic gene

cluster in Saccharopolyspora erythraea. Mal Gen Genet 1997, 256:239-251.

The authors, independently of those in [30°°], report the isolation, sequencing and disruption analysis of the entire gene cluster responsible for the biosynthesis of the deoxysugar components of erythromycin. The proposed sequence of steps in the pathways are very similar, with only slight differences. Together with [30°°], this paper represents a significant leap forward in our understanding of deoxysugar biosynthesis.

30. Summers RG, Donadio S, Stayer M J, Wendt-Pienkowski E, • • Hutchinson CR, Katz L: Sequencing and mutagenesis of genes

from the erythromycin biosynthetic gene cluster of Saccharopolyspora erythraea that are involved in L-mycarose and D-desosamine production. Microbiology 1997, 143:3251-3262.

The authors, independently of those in [29"'], report the isolation, sequencing and disruption analysis of the entire gene cluster responsible for the biosynthesis of the deoxysugar components of erythromycin. The proposed sequence of steps in the pathways are very similar, with only slight differences. Together with [29°°], this paper represents a significant leap forward in our understanding of deoxysugar biosynthesis.

31. Gandecha AR, Large SL, Cundliffe E: Analysis of four tylosin • biosynthetic genes from the tylLM region of the Streptomyces

fradiae genome. Gene 1997, 1 84:197-203. Of the four genes analyzed in this paper, two of them encode a glycosyltransferase (tylM2) and a methyltransferase (tylM1) that are involved in the biosynthesis of mycaminose, one of the deoxysugars in tylosin. A reasonable biosynthetic route to mycaminose is proposed.

32. Gallo MA, Ward J, Hutchinson CR: The dnrM gene in Streptomyces peucetius contains a naturally occurring frameshif t mutation that is suppressed by another locus outside of the daunorubicin- production gene cluster. Microbiology 1996, 142:269-275.

33. Madduri K, Hutchinson CR: Functional characterization and transcriptional analysis of the dnrR 1 locus, which controls daunorubicin biosynthesis in Streptomyces peucetius. J Bacterial 1995, 177:1208-1215.

34. Thorson JS, Lo SF, Liu H-w, Hutchinson CR: Biosynthesis of 3,6- dideoxyhexoses: new mechanistic reflections upon 2,6-dideoxy, 4,6-dideoxy, and amino sugar construction. J Am Chem Sac 1 993, 115:6993-6994.

35. Otten SL, Liu X, Ferguson J, Hutchinson CR: Cloning and characterization of the Streptomyces peucetius dnrQS genes encoding a daunosamine biosynthesis enzyme and a glycosyl transferase involved in daunorubicin biosynthesis, J Bacteria/ 1995, 177:6688-6692.

36. Scotti C, Hutchinson CR: Enhanced antibiotic production by manipulation of the Streptomyces peucetius dnrH and dnm T genes involved in doxorubicin (adriamycin) biosynthesis. J Bacterial 1996, 178:7316-7321.

37. Liu H-w, Thorson JS: Pathways and mechanisms in the biogenesis of novel deoxysugars by bacteria. Annu Rev Microbiol 1994, 48:223-256.

38. Jacobsen JR, Hutchinson CR, Cane DE, Khosla C: Precursor- directed biosynthesis of erythromycin analogs by an engineered polyketide synthase. Science 1997, 277:367-369.

39. Marsden AFA, Wilkinson B, Cort6s J, Dunster N J, Staunton J, Leadlay PF: Engineering broader specificity into an antibiotic- producing polyketide synthase. Science 1 998, 279:199-201.

Deoxysugar b iosynthes is Johnson and Liu 649

40. Solenberg PJ, Matsushima P, Stack DR, Wilkie SC, Thompson PC, • , Baltz RH: Production of hybrid glycopeptide antibiotics in vitro and

in Streptomyces toyocaensis. Chem Biol 1997, 4:1 95-202. Cloned glycosyltransferases from glycopeptide antibiotic producers were used to produce novel hybrid antibiotics, both in vitro and in vivo. This is one of the first reports on the use of sugar biosynthetic genes to produce new compounds, and it underscores the future potential of utilizing glycosyltransferases to create valuable drugs.

41. Madduri K, Kennedy J, Rivola G, Inventi-Solari A, Filippini S, °° Zanuso G, Colombo A L, Gewain KM, Occi JL, MacNeil DJ,

Hutchinson CR: Production of the antitumor drug epirubicin

(4'-epidoxorubicin) and its precursor by a genetically engineered strain of Streptomyces peucetius./Vat Biotechnol 1998, 16:69-74.

A fermentation method for producing 4'-epidoxorubicin is reported in this paper. The method involves cloning heterologous Streptornyces averrnitilis avrE or Saccharopolyspora eryBIV genes into an S. peucetius dnrnV mutant blocked in the biosynthesis of daunosamine. These experiments not only unambiguously assigned AvrE and EryBIV as ketohexose reductases, but they also clearly demonstrated that combinatorial biosynthesis with deoxysugar biosynthetic genes can be used to generate valuable hybrid antibiotics.

42. Stubbe J, van der Donk W: Protein radicals in enzyme catalysis. Chem Rev 1998, 98:705-762.

mechanisms and pathways from recent deoxysugar biosynthesis research

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