enzymology and molecular biology of lignin degradation · 13 enzymology and molecular biology of...

13 Enzymology and Molecular Biology of Lignin Degradation

D. CULLEN1 and P.J. KERSTEN1

CONTENTSI.A.B.II.A.B.C.D.III.IV.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295Lignin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295Microbial Degradation of Lignin . . . . . . . . . . . . 296Enzymology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297Lignin Peroxidase . . . . . . . . . . . . . . . . . . . . . . . . . 297Manganese Peroxidase . . . . . . . . . . . . . . . . . . . . . 300Glyoxal Oxidase . . . . . . . . . . . . . . . . . . . . . . . . . . 301Other Related Enzymes . . . . . . . . . . . . . . . . . . . . 301Molecular Biology of Ligninolytic Fungi . . . . . . 302Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306Reference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306

I. Introduction

Lignin is the most abundant aromatic polymer innature. It is synthesized by higher plants, reachinglevels of 20–30% of the dry weight of woodytissue. Although white-rot fungi were longrecognized as efficient lignin-degrading microbes,research on their enzymology and genetics wassomewhat neglected until the past decade. Theimpetus for increased research interest can betraced to the discovery of “ligninases” and poten-tial commerical applications in the pulp and paperindustry and in the degradation of xenobiotics. Inthis chapter, we briefly summarize several decadesof research on white-rot fungi, and readers arereferred to several comprehensive reviews for ad-ditional background information. Recent devel-opments in molecular biology and enzymology areemphasized. Areas where knowledge is incom-plete are highlighted.

A. Lignin

Lignin is a complex, three-dimensional, non-stereoregular polymer composed of phenylpro-

1 Institute of Microbial Biochemical Technology. ForestProducts Laboratory, US Forest Service, One GiffordPinchot Drive. The University of Wisconsin. Madison. WI53705-2398. USA

panoid units linked through several major types ofcarbon-carbon and ether bonds. Lignin biogenesisinvolves free radical polymerization of theprecusors, p -coumaryl, coniferyl, and sinapylalcohols. the relative proportions depending onthe type of plant and tissue. Plant peroxidasescatalyze the one-electron oxidation of these pre-cursors to generate phenoxy radical intermediateswhich diffuse away from the enzyme and couplewith each other and the growing lignin polymer.This random coupling generates oligomericquinone methides susceptible to nucleophilic at-tack at the benzyl carbons by water, phenolichydroxyls of other lignols, and also by thehydroxyls of hemicellulose to form lignin-carbo-hydrate complexes. The interunit bonds arecharacterized by the points of attachment: thestandard nomenclature designates positions onthe propyl side chains as α, β, γ (α be ingproximal to the aromatic ring) and positionson the aromatic ring as 1–6 (1 indicating thepoint of attachment of the propyl side chain).Thus, the β−0−4 ether bond is quantitatively themost important interunit bond in spruce lignin(Fig. 1).

The bulk of the lignin is in the thick secondarycell walls, but highest lignin concentrations are inthe middle lamellae (intercellular regions), wherethe lignin cements the plant cells together, therebyproviding rigidity and strength to the plant. Rela-tive to cellulose and hemicellulose. which are com-posed of linear, repeating hydrolyzable interunitbonds, lignin represents a formidable microbialsubstrate. Further, the lignin polymer encruststhe cellulosic microfibrils of plants and is che-mically bonded to the hemicelluloses, makingthese polysaccharides less accessible to microbialdecay. Lignin synthesis, structure and chemistryhave been reviewed by Adler (1977), Harkin(1967), Higuchi (1990), and Sarkanen and Ludwig(1971).

The Mycota IIIBiochemistry and Molecular BiologyBrambl/Marzluf (Eds.) Springer-Verlug Berlin Heidelberg 1996

296 D. Cullen and P.J. Kersten

Fig. 1. Structure of lgnin and precursors. An example of a β−0−4 linkage is shown between residues 1 and 2, and a β− 1between residues 8 and 9. (Kirk and Farrell 1987)

B. Microbial Degradation of Lignin

The white-rot basidiomycetes degrade lignin moreextensively and rapidly than any other knowngroup of organisms. In contrast to other fungi andbacteria, white-rot fungi are capable of completelignin degradation to carbon dioxide and water.The species are widely distributed, occurring inboth tropical and temperate environments. White-rot fungi are also well adapted for utilizing otherplant components and the species vary substan-tially with regard to their relative cellulolyticversus ligninolytic efficiency. The microbiology oflignin degradation has been reviewed (Kirk andShimada 1985; Buswell and Odier 1987; Kirk andFarrell 1987; Eriksson et al. 1990).

The ability of many other organisms to de-grade lignin is somewhat uncertain. This is due inpart to the inability of microbes, including thewhite-rot basidiomycetes, to use lignin as sole

source of carbon or energy. Consequently, there isno easy selection for lignin degradation by con-ventional methods. Also, the characterizations ofminor modification and depletion of lignin in na-tive lignocellulose often are equivocal, due to itsintimate association with the other plant constitu-ents. Brown-rot fungi, also basidiomycetes, causerapid and extensive decay of the hemicelluloseand cellulose of wood, leaving a brown modifiedlignin residue (Kirk and Adler 1970).

The most extensively characterized white-rotfungus is Phanerochaete chrysosporium, previ-ously known as Chrysosporium lignorum andSporotrichum pulverulentum (Burdsall and Eslyn1974; Raeder and Broda 1984). Strains vary sub-stantially, but certain isolates are among the mostefficient lignin degraders known. The species iswell suited to laboratory study; it grows rapidlyon defined glucose media, has a high tempera-ture optimum for growth, conidiates profusely,

Lignin Degradation 297

and produces basidiospores under establishedconditions.

Using 14C-lignin as substrate in defined media,culture parameters required by Phanerochaete forlignin degradation to CO2 and H2O have beendetermined. Ligninolysis is triggered by nutrientlimitation; cultures starved for carbon, nitrogen,or sulfur are able to oxidize lignin to CO2 (Keyseret al. 1978; Kirk et al. 1978; Reid 1979; Jeffries etal. 1981). The fungus is typically grown in station-ary liquid medium at pH 4.5, 39°C, high oxygenpartial pressure, and with glucose as the carbonsource and NH4

+ as the limiting nitrogen source.The influence of O2 partial pressure (Kirk et al.1978; Reid 1979; Bar-Lev and Kirk 1981), agita-tion (Kirk et al. 1978; Jäger et al. 1985). metal ionbalance (Jeffries et al. 1981) and pH (Kirk et al.1978) have been studied. Although lignin is notrequired to induce the ligninolytic system, P.chrysosporium synthesizes veratryl alcohol (3,4-dimethoxybenzyl alcohol), which has a lignin aro-matic substituent pattern (Lundquist and Kirk1978; Shimada et al. 1981).

Because of the complexity and heterogeneityof lignin polymers, most detailed studies havebeen in defined media with lignin model com-pounds, e.g., β−0−4 and β−1 lignin substructuremodels. Oxidations of the lignin substructures,e.g., C α - C β cleavage and Ca oxidation (Enoki etal. 1980; Goldsby et al. 1980; Enoki and Gold1982; Nakatsubo et al. 1982; Kirk and Nakatsubo1983; Kamaya and Higuchi 1984; Umezawa andHiguchi 1984; Umezawa and Higuchi 1985), agreewell with those deduced from the studies withlignin polymer (Chen et al. 1982, 1983; Chua et al.1982).

II. Enzymology

A. Lignin Peroxidase

Growing evidence indicates that lignin per-oxidases and manganese peroxidases play a cen-tral role in the initial degradation of the complexaromatic polymer lignin by P. chrysosporium.Lignin peroxidase was first discovered based onthe H2O2-dependent C α - C β cleavage of ligninmodel compounds and subsequently shown tocatalyze the partial depolymerization of methyl-ated lignin in vitro (Glenn et al. 1983; Tien andKirk 1983; Gold et al. 1984; Tien and Kirk 1984).

Since the first reports of lignin peroxidase, severalisozymic forms have been detected with P.chrysosporium and a number of other white-rotfungi. For the purposes of this chapter, theenzymology of lignin biodegradation will be lim-ited principally to those enzymes secreted byP. chrysosporium under ligninolytic conditions.

The principal criteria for identifying specificlignin peroxidase isozymes are their pI and theirorder of elution from a Mono Q anion exchangecolumn (Renganathan et al. 1985; Kirk et al.1986a; Leisola et al. 1987). Ten peroxidases areseparated by Mono Q chromatography and aredesignated H1 through H10. Six of these, H1 (pI4.7), H2 (pI 4.4), H6 (pI 3.7), H7 (pI 3.6), H8 (pI3.5), and H10 (pI 3.3) have veratryl alcohol oxida-tion activity characteristic of lignin peroxidase(Farrell et al. 1989). Analytical isoelectric focusinghas resolved 15 proteins with lignin peroxidaseactivity (Leisola et al. 1987). Growth conditions(e.g., N vs. C starved), purification methods, andstorage can affect relative isozymic levels.

The isozymes of lignin peroxidase areglycoproteins with molecular weights estimated at38–46 kDa. Structural relatedness is evident bythe crossreactivity for the various isozymes withpolyclonal antibodies raised against H8 ligninperoxidase. However, N-terminal and geneticanalyses (see below) show lignin peroxidases to beseparate gene products and not solely due toposttranslational modifications. Spectroscopic andphysical characterizations indicate that ligninperoxidase is a heme protein; the iron of nativeenzyme is high-spin and pentacoordinate, withhistidine as the fifth ligand (Kuila et al. 1985;Andersson et al. 1987; Banci et al. 1991). Primarysequence deduced from DNA clones also predictssimilarities with other well-characterized peroxi-dases such as horseradish peroxidase and cyto-chrome c peroxidase (see below).

Enzyme intermediates in the catalytic cycleof lignin peroxidase are analagous to otherperoxidases (e.g, horseradish peroxidase); steady-state and transient-state kinetics have been stud-ied in detail (Andrawis et al. 1988; Harvey et al.1989; Marquez et al. 1988; Renganathan and Gold1986; Tien et al. 1986; Wariishi et al. 1990). H2O2

is the preferred co-substrate and oxidant in thelignin peroxidase-catalyzed reactions. The inter-action of lignin peroxidase with its substrates is bya ping-pong mechanism, i.e., H2O2 oxidizes ferricenzyme by two electrons to give CompoundI intermediate (one oxidizing equivalent as an


oxyferryl center and the other in the porphyrincation radical); Compound I oxidizes aromaticsubstrates by one electron to give Compound II(a one-eletron oxidized enzyme intermediate),which again oxidizes aromatic substrates to returnthe enzyme to resting state. Although the variouslignin peroxidases have similar specificities to-wards aromatic substrates (Farrell et al. 1989:G1umoff et al. 1990), their kinetic behaviour variesmore clearly when tert -butyl hydroperoxide is sub-stituted for H2O2 (Glumoff et al. 1990). An unu-sual characteristic of lignin peroxidase is its lowpH optimum (< pH 3), even though there is no pHdependence for Compound I formation. Conver-sion of catalytic intermediates are depicted belowwith S representing a nonphenolic aromaticsubstrate.

Native (ferric) peroxidase + H2O2

+ Compound I + H2O

Compound I + S + Compound II + S+

Compound II+ S + Native (ferric)peroxidase + S+

Another well-characterized peroxidase en-zyme intermediate is Compound III. This inter-mediate is formed by complex formation of(i) oxygen with the ferrous peroxidase (nativeperoxidase reduced by one electron), (ii)superoxide with the ferric peroxidase, and (iii)H2O2 with Compound II. With lignin peroxidase,there is conflicting evidence as to the stability oflignin peroxidase Compound III, and whetherCompound III* (a complex of lignin peroxidaseCompound III with H2O2) is a true enzyme inter-mediate (Cai and Tien 1989, 1990, 1992; Wariishiand Gold 1990; Wariishi et al. 1990; Wariishi et al.1992). Discrepancies may be a result of the insta-bility of lignin peroxidase with storage (thus gen-erating a heterogeneous population of enzymeintermediates), the use of experimental conditionsthat may not be valid with lignin peroxidase (e.g.,in single-turnover experiments and in the detec-tion of superoxide), and isozymic differences (Caiand Tien 1992).

The crystal structure of lignin peroxidase isnow known (Edwards et al. 1993; Pointek et al.1993). Most striking is the similarity of the overallthree-dimensional structure to that of cytochromec peroxidase (CCP), even though sequence iden-tity is only approximately 20%. In both cases, theproximal heme ligand is a histidine that is hydro-

gen bonded to a buried aspartic acid residue; theperoxide pocket is also similar with distal histidineand arginine. In contrast to CCP, which hastryptophans contacting the distal and proximalheme surfaces, lignin peroxidase has phenyla-lanines. Furthermore, the hydrogen bonding ofthe heme propionate of lignin peroxidase to Asp-183 (in contrast to Asn with CCP) may explain thelow pH optimum of lignin peroxidase (Edwards etal. 1993). Other notable features of ligninperoxidase are four disulfide bonds (CCP hasnone) and a C -terminal region without regularextended secondary structure (residues 276 to343), for which there is no equivalent in CCP.

Lignin peroxidase catalyzes a variety of oxida-tions, all of which are dependent on H2O2 Theseinclude C α - C β cleavage of the propyl side chains oflignin and lignin models, hydroxylation of benzylicmethylene groups, oxidation of benzyl alcohols tothe corresponding aldehydes or ketones, phenoloxidation, and even aromatic ring cleavage ofnonphenolic lignin model compounds (Tien andKirk 1984; Leisola et al. 1985; Renganathan et al.1985; Umezawa et al. 1986; Umezawa and Higuchi1987). Although the assortment of reactions isvery complex, the initiation of these reaction issimple. Lignin peroxidase oxidizes the aromaticsubstrates by one electron (Fig. 2); the resultingaryl cation radicals degrade spontaneously viamany reactions dependent on the structure of thesubstrate and on the presence of reactants (Fig. 3).Production of cation radical intermediates frommethoxybenzene substrates was detected by ESR(Kersten et al. 1985) in reactions catalyzed bylignin peroxidase. Using more lignin-related com-pounds, Hammel et al. (Hammel et al. 1986b) fur-ther proved the involvement of radicalintermediates by identifying radical-dimer prod-ucts, as well as carbon-centered and peroxyl radi-cal intermediates. The central role of reactivecation radical intermediates was also proposed byothers and tested with che-mical one-electronoxidants (Harvey et al. 1985; Shoemaker et al.1985).

Although the reactions subsequent to cationradical formation are complex, they follow chemi-cal principles that readily explain the predominantproducts. Thus the C α - C β cleavage (Fig. 3) that ispredominant with model compounds most closelyrelated to lignin (Hammel et al. 1986b; Kirk et al.1986b) can be understood based on the degreeof substitution of the C β carbon (Snook and


Fig. 2. Formation of aryl cation radical intermediate by theoxidation of substrate by one electron

Fig. 3. C α - C β cleavage and C α oxidation as a result of spon-taneous reactions subsequent to aryl cation radical forma-tion. (After Kirk et al. 1986b)

Hamilton 1974). The cation radical mechanismalso explains the oxygenase properties of ligninperoxidase; O2 reacts with carbon-centered radicalintermediates that result from C-C bond cleav-age of cation radicals (Hammel et al. 1985;Renganathan et al. 1986). Lignin peroxidase activ-ity is also expected to break the C α lignin-carbo-hydrate bond (Kirk et al. 1986b). Detailed reviewson the radical chemistry of lignin peroxidase-catalyzed reactions are provided elsewhere(Higuchi 1990; Shoemaker 1990).

To complete the peroxidative catalytic cycle,lignin peroxidase Compound I and CompoundII must be sufficiently positive to remove oneelectron from nonphenolic substrates. Hammel etal. (1986a) demonstrated that lignin peroxidaseoxidizes polycyclic aromatics with ionizingpotentials of approximately 7.5eV or lower,whereas horseradish peroxidase will oxidize onlythose polycyclics with ionization potentials below7.35eV (Cavalieri and Rogan 1985). Similarly,the homologous series of methoxybenzenes(half-wave potentials range from 0.81 V to 1.76 V

vs. a saturated calomel electrode) has been usefulin characterizing the relative oxidation potentialsof oxidative enzymes. In the presence of H2O2,lignin peroxidase oxidizes ten with the lowesthalf-wave potentials, whereas horseradish per-oxidase oxidizes the four lowest, and laccase (withO2 as electron acceptor) oxidizes only the lowest(Kersten et al. 1990). The direct measure ofthe ferric-ferrous couple of lignin peroxidaseindicates that the heme active site is more elec-tron-deficient than other peroxidases, and thuswould predict that the catalytic enzymic interme-diates are of higher redox potential (Millis et al.1989).

The ability of catalytic amounts of goodsubstrates for lignin peroxidase (e.g., veratryl al-cohol) to greatly enhance the oxidation of poorsubstrates (e.g., anisyl alcohol) was demonstratedby Harvey et al. (Harvey et al. 1986). One expla-nation for this stimulation is that the cation radicalintermediates generated from the substrates canmediate oxidations of poor substrates. Further-more, the veratryl alcohol may play an importantrole in protection of the peroxidase during itscatalytic cycle. and thus also contribute to theobserved stimulation (reviewed by Shoemaker1990). The mediation by cation radical intermedi-ates is also likely in the oxidation of oxalic acid(Akamatsu et al. 1990; Popp et al. 1991) and H2O2

(Barr et al. 1993). With oxalate, veratryl alcohol,and Mn2+, the catalysis with lignin peroxidase pro-ceeds with the consumption of oxygen and gen-eration of carbon dioxide anion radicals andperhydroxyl radicals (Popp et al. 1991). Oxalate isalso proposed to have a role in preventing ligninperoxidase Compound III formation (Goodwinet al. 1994).

Lignin peroxidase also catalyzes the oxidationof phenolics. In regard to the mechanism of lignindepolymerization, these reactions are of interestbecause phenolics are produced by the action oflignin peroxidase on nonphenolic lignin struc-tures. This would explain both the depolymeri-zation of lignin (Tien and Kirk 1983) and also therepolymerization of lignin fragments in vitro(Haemmerli et al. 1986: Odier et al. 1988). Morerecently, the partial depolymerization of a syn-thetic lignin with crude lignin peroxidase andH2O2 has been demonstrated in vitro (Hammeland Moen 1991). Dilute lignin dispersions and lowsteady-state H2O2 concentration are thought to beimportant in minimizing bimolecular coupling of


phenoxy radicals that would lead to polymeriza-tion in vitro (Hammel and Moen 1991). It is alsoproposed that glycosylation of lignin break-down products may be important in favoring thedepolymerizing reactions (Kondo et al. 1990).

The importance of lignin peroxidase indepolymerization of lignin in vivo was convinc-ingly demonstrated by Leisola et al. (1988). Addi-tion of exogenous lignin peroxidase to carefullywashed mycelial pellets greatly stimulated theconversion of 14C-lignin to 14CO2. When veratrylalcohol was added, a further stimulator effectwas observed. Horseradish peroxidase had noeffect. These results suggest that the presence ofmycelia may play an important role in favoringoverall depolymerization by removing lignin frag-ments as they are released. Likewise, Kurekand Odier have shown that extracellular ligninperoxidase stimulates the mineralization of ligninin cultures at levels up to 20nkat ml-1 and thatlignin peroxidase localized on the surface of themycelia is most effective (Kurek and Odier 1990).

B. Manganese Peroxidase

Another heme peroxidase found in theextracellular fluid of ligninolytic cultures ofP. chrysosporium is manganese peroxidase(Kuwahara et al. 1984; Paszczynski et al. 1985).The principle function of the enzyme is to oxidizeMn2+ to Mn3+, using H2O2 as oxidant. Activity ofthe enzyme is stimulated by simple organic acidswhich stabilize the Mn3+, thus producing diffusibleoxidizing chelates (Glenn and Gold 1985; Glenn etal. 1986). Recently, physiological levels of oxalatein P. chrysosporium cultures have been shown tostimulate manganese peroxidase activity (Kuanand Tien 1993).

As with lignin peroxidase, the prostheticgroup is iron protoporphryn IX and severalisozymes can be detected (Paszczynski et al. 1986;Leisola et al. 1987; Mino et al. 1988; Wariishi et al.1988). The 46-kDa glycoproteins do not crossreactwith polyclonals raised against lignin peroxidase,and the peptide mapping patterns are differentfrom those observed with lignin peroxidase(Leisola et al. 1987). Consistent with the nomen-clature used for the lignin peroxidase isozymes(Farrell et al. 1989), specific manganese peroxi-dases identified in cultures are H3 (pI = 4.9),H4 (pI = 4.5), and H5 (pI = 4.2) (Pease and Tien1992).

Manganese peroxidase enzyme intermediateare analagous to other peroxidases (Wariishi et a1988, 1989). Native manganese peroxidase is oxi-dized by H2O2 to Compound I, which can then bereduced by Mn2+ and phenols to generate Com-pound II. Compound II then is reduced back to aresting state by Mn2+, but not by phenols (Wariishet al. 1989). Therefore Mn2+ is necessary to com-plete the catalytic cycle and shows saturation ki-netics (Wariishi et al. 1988; Pease and Tien 1992Kinetic studies with Mn2+ chelates supportrole for oxalate in the reduction of manganeseperoxidase Compound II by Mn2+ (Kuan et a1993; Kishi et al. 1994).

Native (ferric) peroxidase + H2O2

+ Compound I + H2O

Compound I + Mn2+ + Compound II + Mn3+

Compound I + AH + Compound II+ A · + H+

Compound II+ Mn2+ + Native (ferric)peroxidase + Mn3+

The crystal structure of manganeseperoxidase shows similarities with ligninperoxidase; the active site has a proximal Hisligand H-bonded to an Asp, and a distal side per-oxide-binding pocket consisting of a catalytic Hisand Arg (Sundaramoorthy et al. 1994). Howeverthere is also a proposed manganese-binding siteinvolving Asp-179, Glu-35, Glu-39, and a hemepropionate. In contrast to lignin peroxidase, man-ganese peroxidase has five disulfide bonds.

Typical assays for monitoring manganeseperoxidase activity use phenolics that are easilyoxidized by Mn3+ and that give useful colorchanges for detection. However, there has beensome question as to the role of the enzyme inlignin depolymerization. The biomimetic oxida-tion of lignin model compounds by Mn3+ suggeststhat it may play a role in oxidizing both phenolicand nonphenolic residues of lignin (Hammel et al.1989). More recently, the in vitro partial de-polymerization of synthetic lignin by manganeseperoxidase has been demonstrated (Wariishi et al.1991).

C. Glyoxal Oxidase

An important component of the ligninolytic sys-tem of P. chrysosporium is the H2O2 that is re-quired as oxidant in the peroxidative reactions. A


number of oxidases have been proposed to play arole in this regard. However, the only one thatappears to be secreted in this system under stand-ard ligninolytic conditions is glyoxal oxidase (Kirkand Farrell 1987).

The temporal correlation of glyoxal oxidase,peroxidase, and oxidase substrate appearances incultures suggests a close physiological connectionbetween these components (Kersten and Kirk1987; Kersten 1990). The oxidase is a glycoproteinof 68 kDa with two isozymic forms (pI 4.7 and 4.9).The active site of the enzyme has not been charac-terized, but Cu2+ appears to be important inmaintaining activity of purified enzyme. Glyoxaloxidase is produced in cultures when P .chrysosporium is grown on glucose or xylose, themajor sugar components of lignocellulosics. Thephysiological substrates for glyoxal oxidase, how-ever, are not these growth-carbon compounds, butapparently intermediary metabolizes. A numberof simple aldehyde-, α -hydroxycarbonyl-, and α−dicarbonyl compounds are substrates.

Lignin itself is a likely source of glyoxaloxidase substrates. Oxidation of a β−0−4 modelcompound (representing the major substruc-ture of lignin) by lignin peroxidase releasesglycolaldehyde (Hammel et al. 1994). Glycolal-dehyde is a substrate for glyoxal oxidase andsequential oxidations yield oxalate and multipleequivalents of H2O2. The oxalate may, in turn, bea source of chelate required for the manganeseperoxidase reactions described above.

The reversible inactivation of glyoxal oxidaseis a property perhaps of considerable physiologi-cal significance (Kersten 1990; Kurek and Kersten1995). Glyoxal oxidase becomes inactive duringenzyme turnover in the absence of a coupledperoxidase system. The oxidase is reactivated,however, by lignin peroxidase and non-phenolicperoxidase substrates. Conversely, phenolics pre-vent the activation by lignin peroxidase. Thissuggests that glyoxal oxidase has a regulatorymechanism that is responsive to peroxidase,peroxidase substrates, and peroxidase products(e.g., phenolics resulting from ligninolysis). Nota-bly, lignin will also activate glyoxal oxidase in thecoupled reaction with lignin peroxidase.

D. Other Related Enzymes

Unlike many white-rot fungi, Phanerochaete hasno detectable laccase activity. Recent evidence,

however, suggests that laccase, like lignin per-oxidase, plays a role in lignin degradation by fungi(Morohoshi et al. 1987; Kawai et al. 1988, 1989).The laccase of Trametes (syn. Coriolus o rPolyporus) versicolor is a blue copper oxidase thatcatalyzes the four-electron reduction of O2 to H2Oduring its oxidation of phenolics, aromatic amines,ascorbate and metal cyanides (Malmström et al.1975). As with lignin peroxidase and manganeseperoxidase, oxidation of phenolics by laccase un-dergo further nonenzymatic reactions. Laccasehas four copper atoms with type-1 and type-3 cop-per sites of particularly high redox potentialin comparison to other blue copper proteins(Reinhammer 1984). The laccase of Phlebiaradiata is reported to have only two coppers,in conjunction with pyrroloquinoline quinone(PQQ) in the active site (Karhunen et al. 1990).However, PQQ is not detected in the laccase ofTrametes versicolor (Maccarrone et al. 1991). Thesynergism between the laccase and manganeseperoxidase of Rigidopporus lignosus is an exampleof the possible complex relationships that may beimportant in lignocellulose degradation (Gallianoet al. 1991).

Cellobiose oxidase (Ayers et al. 1978) andcellobiose: quinone oxidoreductase (CBQase)(Westermark and Eriksson 1974) may be involvedin both lignin and cellulose degradation. Limitedproteolysis of cellobiose oxidase indicates thatCBQase is probably a breakdown product(Henriksson et al. 1991; Wood and Wood 1992).Cellobiose oxidase has two domains, one contain-ing a flavin and the other containing a heme. Theflavin-containing domain binds cellulose and isfunctionally similar to CBQase. A role proposedfor these oxidoreductases is to prevent repoly-merization of phenoxyradicals produced by per-oxidases and laccases during lignin oxidation(Eriksson et al. 1993). The more recent anddiverse observations with the oxidoreductasessuggest multiple roles for the enzymes, e.g.. inelectron-transfer reactions with cytochromes(Rogers et al. 1994), quinone reduction, manga-nese oxide solubilization, and in Mn3+ chelation(Roy et al. 1994).

Although the peroxide-generating enzymepyranose oxidase (synonym, glucose-2-oxidase) ispredominantly intracellular in liquid cultures of P.chrysosporium, there is evidence that the oxidaseplays an important role in wood decay (Daniel etal. 1994). The oxidase is preferentially localized inthe hyphal periplasmic space and the associated


membranous materials. Similar ultrastructuraldistribution is observed with manganese peroxi-dase, suggesting a cooperative role. Another strat-egy for peroxide generation is observed withBjerkandera sp. strain BOS55 which producesextracellular aryl alcohol oxidase (de Jong et al.1994). The preferred substrates are chlorinatedanisyl alcohols which the fungus synthesizes denovo from glucose. The oxidation products arereduced by the fungal mycelium, presumably byan aryl alcohol dehydrogenase, and thus recycled.An aryl alcohol dehydrogenase has been purifiedand characterized from P. chrysosporium(Muheim et al. 1991).

III. Molecular Biologyof Ligninolytic Fungi

Knowledge concerning the molecular genetics ofthe P. chrysosporium ligninolytic system has ad-vanced considerably in the past few years.Standard procedures have been established forauxotroph production (Gold et al. 1982), recombi-nation analysis (Alic and Gold 1985), and rapidDNA and RNA purification (Haylock et al. 1985;Raeder and Broda 1985). Aspects of the molecu-lar biology of P. chrysosporium have been re-cently reviewed (Alic and Gold 1991; Pease andTien 1991; Cullen and Kersten 1992; Gold andAlic 1993).

Several groups have developed transforma-tion systems for P. chrysosporium. Randall andcoworkers (1989) reported transformation basedon resistance to the amino glycoside antibiotic,G418. Resistance was conferred by the Kanr deter-minant of Tn903. This system appears to involverecombination of the Kan r vector with anendogenous plasmid of P. chrysosporium strainME446. The recombinant plasmids are main-tained extrachromosomally at low copy numbers(Randall and Reddy 1991, 1992; Randall et al.1991). In contrast, a system based on resistance tothe glycopeptide, phleomycin, involves stable in-tegration of the vector into the genome (Raederand Gessner 1992).

Adenine auxotrophs of P. chrysosporiumhave been transformed with the correspondingwild-type gene from Schizophyllum commune(Alic et al. 1989, 1990) and from P. chrysosporium(Alic et al. 1991). These transformations involved

stable integration of the complementing vectorsinto the genome. Recent studies targeted genereplacements to the wra3 locus (Alic et al. 1993)thereby demonstrating homologous recombina-tion in P. chrysosporium. The technique providesa powerful experimental approach for investigat-ing gene function.

Since Tien and Tu (1987) first reported clon-ing of the cDNA encoding lignin peroxidase H8,much has been learned concerning the number,structure, and organization of the peroxidasegenes of P. chrysosporium. It is now clear thatlignin peroxidases are encoded by a family of atleast ten closely related genes and their allelicvariants, and that many of these genes are linked.Unfortunately, the nomenclature designatingclones/genes has not been uniform. Further com-plicating the situation, allelic relationships havenot been established among all clones, and severaldifferent P. chrysosporium strains are now in wideuse. A uniform nomenclature was recently pro-posed for the ten P. chrysosporium genes, whichare designated lipA through lipJ (Gaskell et al.1944).

Extreme sequence conservation among cer-tain clones and the presence of allelic forms en-coding slightly different isozymes (Gaskell et al.1991) have caused difficulties in identifyingprecise gene-isozyme relationships. Identificationsmade solely by matching deduced amino acid se-quence with short regions of experimentally deter-mined sequence should be viewed as tenuous.Nevertheless, on the basis of experimentally de-termined isoelectric points, N-terminal sequencedata, and transcript abundance, it is probable thatlipA, lipD, and lipC encode isozymes H8, H2, andH10, respectively. The identity of lipA was sub-stantiated further by heterologous expression ofthe isozyme (Johnson and Li 1991). Glumoff et al.(1990) purified five lignin peroxidase isozymesfrom carbon starved cultures and determined theirN-terminal amino acid sequences. On this basis,three isozymes with pIs of 4.65,3.85, and 3.70 canbe tentatively identified as products of lipD, lipAand lipB, respectively.

Relatively little is known concerning theperoxidase genes of other fungal species. Cloneshave been sequenced from Phlebia radiata(Saloheimo et al. 1989), Trametes versicolor(Black and Reddy 1991; Jonsson and Nyman1992), and Coprinus cinereus (Andersen et al1992). On the basis of Southern blot hybridization


to the P. chrysosporium genes, lignin peroxidase-like sequences are present in the genomes ofBjerkandera adjusta, Coriolus versicolor, Fomeslignosus (Huoponen et al. 1990), Ceriporiopsissubvermispora, and Phlebia brevispora (Ruttimanet al. 1992).

Nucleotide and amino acid sequence homol-ogy is high among the lignin peroxidase genes(Fig. 4). For example, P. chrysosporium strainBKM-1767 genes lipA and lipI encode proteinswhich are 97% identical (Schalch et al. 1989).Residues believed essential to peroxidase activityare conserved (Fig. 4, arrows 3, 4, 5). The lipAgene also features a putative propeptide (residues22–28; Schalch et al. 1989). A similar sequencewas shown in a strain OGC1O1 gene, and theproenzyme was identified as an in vitro translationproduct (Ritch et al. 1991). Many of the P.chrysosporium genes feature a proline-richcarboxy terminus (Fig. 4), although its significanceis unknown. The P. chrysosporium genes also con-tain eight to nine short introns and the positionsof six of these are highly conserved (Brownet al. 1988; Schalch et al. 1989; Ritch and Gold1992).

Recently, an unusual insertion/mutation wasdetected in lipI (Gaskell et al. 1995). Specifically, a17476p transposon-like element was identifiedimmediately adjacent to the fourth intron of lipI2.The element, designated Pcel, is inherited in asimple 1:1 Mendelian fashion and transcrip-tionally inactivates lipI2. Southern blots revealedthe presence of related sequences in severalstrains, although the copy number was consist-ently low. Transposon-like features include in-verted terminal repeats and a putative TA targetduplication. The sequence showed no similarity toknown transposons. The significance of Peel ingenerating ligninolytic variation in certain P.chrysosporium strains remains uncertain.

Relative to lignin peroxidases, less is knownconcerning the number and structure of the man-ganese peroxidase genes. A cDNA encoding man-ganese peroxidase (Pribnow et al. 1989) and itscorrespondirig genomic clone (Godfrey et al.1990) have been isolated from strain OGC101. Amanganese peroxidase cDNA, MP-1, derivedfrom strain BKM-1767 has also been character-ized (Pease et al. 1989). Amino acid sequence ofthe latter is 50–60% identical to lipA. Additionalclones have been isolated from strains OGC101(Mayfield et al. 1990) and BKM-1767 (Orth et al.

1994), although the former is probably an allelicvariant of MP-1. The total number of manganeseperoxidase genes remains to be established. How-ever, N-terminal amino acid sequence of isozymes(Datta et al. 1991; Pease and Tien 1992) haveshown that at least three manganese peroxidasegenes are expressed in P. chrysosporium strainBKM-1767. As with the lignin peroxidases, man-ganese peroxidase-like sequences have been de-tected in the genomes of C. subvermispora and P.brevispora using heterologous probes (Ruttimanet al. 1992).

Genetic segregation analyses, pulsed-fieldelectrophoresis, and restriction maps of genomicclones have shown that the lignin peroxidasegenes are linked. Raeder and coworkers (1989)demonstrated two linkage groups in their RFLPmap of P. chrysosporium strain ME446. These re-sults agree with Clamped Homogeneous Electri-cal Field (CHEF) electrophoresis (Gaskell et al.1991; Stewart et al. 1992). Three lignin peroxidasegenes (lipA, lipB, lipC) are clustered within a30kb region (Gaskell et al. 1992; Huoponen et al.1990). Recently, PCR amplification and allele-specific probes have permitted segregation analy-ses for all known P. chrysosporium genes (Gaskellet al. 1994). On this basis, the ten lignin peroxidasegenes were assigned to three linkage groups, andeight genes were closely linked. Southern blotanalysis of CHEF gels have localized two manga-nese peroxidase genes to chromosomes separatefrom each other and from the lignin peroxidasegenes (Orth et al. 1994).

In P. chrysosporium strain BKM-1767, ligninperoxidase-containing chromosomes were shownto be dimorphic with respect to migration onCHEF gels. Chromosome bands at 3.5 and 3.7 mbhybridize to eight lignin peroxidases and lipD hy-bridizes to 4.4 and 4.8-mb bands (Gaskell et al.1991; Stewart et al. 1992). During meiosis, the ho-mologous chromosome pairs recombine and seg-regate into single basidiospore (homokaryotic)derivatives, which can be analyzed for the pres-ence of specific alleles (Gaskell et al. 1992).Homologous chromosomes differing in electro-phoretic mobility have also been observed in theprotozoan Plasmodium falciparum (Corcoran etal. 1986, 1988), in S. cerevisiae (Ono and Ishino-Arao 1988), in Candida albicans (Rustehenko-Bulgac et al. 1990), in U. maydis (Kinscherf andLeong 1988) and in C. cinereus (M. Zolan pers.comm.). In P. falicurum. polymorphisms are

304 D.Cullen and P.J. Kersten

111111111

LiPALiPBLiPCLiPDLiPEL i P FLiPGLiPHL i P IL i P J

L i P AL i P BL i P CL i P DL i P EL i P FLiPGLiPHL i P IL i P J

L i P AL i P BL i P CL i P DLiPEL i P FL i P GL i P HL i P I

L i P AL i P BL i P CL i P DL i P EL i P FL i P GL i P HL i P I

Fig. 4. Alignment of P. chrysosporium lignin peroxidase 5), the putative (Schalch et al. 1989; Ritch et al. 1991genes by the Jotun-Hein algorithm (Hein 1990). Vertical secretion signal/propeptide junction (arrow 1), and thearrows indicate conserved active-site residues (arrows 3, 4, propeptide/mature protein junction (arrow 2)


caused by homologous recombination in distal re-gions of chromosomes, and it has been suggestedthat recombination in these dynamic regions maybe related to antigenic variation (Corcoran et al.1988). Translocations have been shown to con-tribute to length polymorphisms in C. albicans(Thrash-Bingham and German 1992). Extensivemapping of chromosome homologies will berequired to determine if a similar mechanism isoperative in P. chrysosporium.

Studies of peroxidase expression have beencomplicated by difficulties in distinguishing tran-scripts of closely related genes, but progress hasbeen made. Several studies have shown that thelignin peroxidase transcription is dramaticallymodulated by culture conditions. Holzbaur andTien (Holzbaur and Tien 1988) examined tran-script levels of the genes encoding isozymes H8(lipA) and H2 (lipD) by Northern blot hybridiza-tion. Under carbon limitation, lipD transcriptsdominated and lipA transcripts were not detected.Under nitrogen limitation, H8 was the most abun-dant transcript and H2 expression was relativelylow. Quantitative RT-PCR techniques (Stewart etal. 1992) and nuclease protection assays (Reiser etal. 1993) were developed to detect all knownlignin peroxidase transcripts. The genes lipA, lipB,and lipI were expressed at similar levels in both C-and N-limited cultures. In contrast, lipC and lipJtranscript levels were substantially increased un-der N-limitation. Transcripts of lipD, and to alesser extent lipE, were abundant under C-limita-tion. Most recently, the quantitative RT-PCRmethod has been extended to complex substratessuch as P. chrysosporium- colonized soil (Lamar etal. 1995).

Several studies have implicated cAMP in-volvement in regulating the lignin degradingsystem of P. chrysosporium. MacDonald andcoworkers (1984, 1985) have shown elevatedintracellular cAMP levels in ligninolytic cultures.Boominathan and Reddy (1992) observed de-creased peroxidase isozymes and transcripts incultures treated with cAMP inhibitors.

Manganese peroxidase production is de-pendent upon Mn concentration (Bonnarme andJeffries 1990; Brown et al. 1990). Regulation byMn is clearly at the transcriptional level (Brownet al. 1990, 1991). Potential transcriptional con-trol elements have been identified in the 5'-untranslated region of the manganese peroxidasegene from strain OGC101 (Godfrey et al. 1990;A1ic and Gold 1991). In addition to Mn(II), tran-

scription is induced by heat shock (Brown et al.1993), hydrogen peroxide, chemical stress, andmolecular oxygen (Li et al. 1995). Pease and Tien(1992) showed differential regulation of manga-nese peroxidase isozymes in C- and N-limited cul-tures. Each isozyme also responded diffently toMn concentration (Pease and Tien 1992). Moukhaet al. (1993) localized manganese peroxidasetranscripts in P. chrysosporium colonies in agar-medium.

The differential regulation of lignin peroxi-dase and manganese peroxidase genes suggestsdifferent functions for the isozymes. Elucidatingthe role and interaction of individual isozymes inlignin degradation will require further biochemi-cal characterization of pure isozymes and theidentification of specific isozymes in complexsubstrates. Enzyme yields from solid wood sam-ples are too low for precise isozyme identification,although immunogold labeling experiments haveidentified lignin peroxidase and manganeseperoxidase in colonized wood (Blanchette et al.1989; Daniel et al. 1990). Recently, Datta et al.(1991) identified manganese peroxidase, ligninperoxidase, and glyoxal oxidase in aspen pulpcolonized by P. chrysosporium. Highly sensitiveRT-PCR techniques (Lamar et al. 1995) may beadapted to detect transcripts of specific genes inwood samples.

An important consideration in these investi-gation must be strain variation. In contrast to P.chrysosporium strain BKM-1767, lipD is the onlylignin peroxidase gene expressed under “stand-ard” ligninolytic conditions (N-limited) in strainME446 (James et al. 1992). Similarly. LiP2 tran-scripts appear to dominate in strain OGC101(Ritch et al. 1991). Clearly, the role and interac-tion of individual genes in lignin degradation mayvary substantially among strains.

Peroxidases are needed for a variety ofbiochemical investigations, but yields of purifiedisozymes from fungal cultures are low andheterologus expression has been problematic.In E. coli, relatively low levels of aggregatedapoprotein are produced in inclusion bodies. At-tempts to recover and reconstitute active ligninperoxidase from E. coli have had limited success(see review by Pease and Tien 1991), althoughactive horseradish peroxidase, a related enzyme,can be recovered from inclusion bodies (Smithet al. 1990). S. cerevisiae expression systems alsoyield little or no intracellular apoprotein (Peaseand Tien 1991). Further, expression of the P.


radiata lignin peroxidases in T. reesei is limited to pulping (Wegner et al. 1991; Kirk et al. 1992),transcripts, even when placed under the control of enzymatic bleaching of pulps (Eriksson andthe highly-expressed and inducible cbhl promoter Kirk 1985), and biodegradation of xenobiotics(Saloheimo et al. 1989). (Hammel 1992; Lamar 1992). In all cases,

Several recent advances have been made fundemental investigations of the underlyingwith regard to heterologous expression of theperoxidases. Baculovirus systems have beenused to produce active recombinant manganeseperoxidase isozyme H4 (Pease et al. 1991) andlignin peroxidase isozyme H8 (Johnson andLi 1991). Although yields are relatively low,baculovirus production may be useful for experi-ments requiring limited quantities of recombi-nant protein, e.g., site-specific mutagenesis). Incontrast, highly efficient secretion of active C.cinereus peroxidase has been demonstrated inAspergillus oryzae (Andersen et al. 1992). Expres-sion was under the control of the A. oryzae Takaamylase promoter, and like the baculovirus sys-tem, addition of herein to the cultures increasedyield substantially.

In contrast to P. chrysosporium peroxidases,glyoxal oxidase is encoded by a single gene(Kersten and Cullen 1993). Although coordinatelytranscribed with the peroxidases (Stewart et al.1992; Kersten and Cullen 1993), the glyoxaloxidase gene is unlinked to all known P. chryso-sporium genes (Gaskell et al. 1994). A cDNA en-coding glyoxal oxidase is efficiently expressed inA. nidulans, yielding a fully active extracellularprotein (Kersten and Cullen, unpubl.).

Little is known concerning the molecular ge-netics of laccase-producing basidiomycetes. Bothgenomic and cDNA clones of Coriolus hirsutus(Kojima et al. 1990) and P. radiata (Saloheimo etal. 1991) have been sequenced. Their deducedamino acid sequences are similar (63%) but di-verge substantially from the laccases of theascomycetous fungi A. nidulans and Neurosporacrassa. The C. hirsutus and P. radiata cDNAs wereexpressed in S. cerevisiae (Kojima et al. 1990) andT. reesei (Saloheimo and Niku-Paavola 1991),respectively. In both cases, active, extracellularenzyme was obtained.

IV. Conclusions

In addition to their key role in carbon cycling,ligninolytic fungi are important in several emerg-ing technologies. Examples include biomechanical

mechanism(s) are needed to develop rationalexperimental approaches toward processimprovement.

Although research has been hampered bysubstrate complexity and by the multiplicity ofenzymes, much progress has been made. Recentlydeveloped experimental tools can be expected tofacilitate major advances in the near future. Forexample, genetic transformation systems allowinggene disruption/replacement may be used to iden-tify the role of specific genes in lignin degradation.Similarly, heterologous expression of peroxidasesand crystal structure determinations may pave theway for site-specific mutagenesis experiments.Thus, fundamental questions concerning struc-ture/function may be addressed and lead tobiotechnical and environmental applications.

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