A series of crystal structures of a meta-cleavage product hydrolase from Pseudomonas fluorescens IP01 (CumD) complexed with various cleavage products

Meta-cleavage product hydrolase (MCP-hydrolase) is one of the key enzymes in the microbial degradation of aromatic compounds. MCP-hydrolase produces 2-hydroxypenta-2,4-dienoate and various organic acids, according to the C6 substituent of the substrate. Comprehensive analysis of the substrate specificity of the MCP-hydrolase from Pseudomonas fluorescens IP01 (CumD) was carried out by determining the kinetic parameters for nine substrates and crystal structures complexed with eight cleavage products. CumD preferred substrates with long non-branched C6 substituents, but did not effectively hydrolyze a substrate with a phenyl group. Superimposition of the complex structures indicated that benzoate was bound in a significantly different direction than other aliphatic cleavage products. The directions of the bound organic acids appeared to be related with the k(cat) values of the corresponding substrates. The Ile139 and Trp143 residues on helix alpha4 appeared to cause steric hindrance with the aromatic ring of the substrate, which hampers base-catalyzed attack by water.     

Substrate specificity of cyclic nucleotide phosphodiesterase from beef heart and from Dictyostelium discoideum

The substrate specificity of beef heart phosphodiesterase activity and of the phosphodiesterase activity at the cell surface of the cellular slime mold Dictyostelium discoideum has been investigated by measuring the apparent Km and maximal velocity (V) of 24 derivatives of adenosine 3',5'-monophosphate (cAMP). Several analogs have increased Km values, but unaltered V values if compared to cAMP; also the contrary (unaltered Km and reduced V) has been observed, indicating that binding of the substrate to the enzyme and ring opening are two separate steps in the hydrolysis of cAMP. cAMP is bound to the beef heart phosphodiesterase by dipole-induced dipole interactions between the adenine moiety and an aromatic amino acid, and possibly by a hydrogen bond between the enzyme and one of the exocyclic oxygen atoms; a cyclic phosphate ring is not required to obtain binding. cAMP is bound to the slime mold enzyme via a hydrogen bond at the 3'-oxygen atom, and probably via a hydrogen bond with one of the exocyclic oxygen atoms. A cyclic phosphate ring is necessary to obtain binding to the enzyme. A specific interaction (polar or hydrophobic) between the base moiety and the enzyme has not been demonstrated. A negative charge on the phosphate moiety is not required for binding of cAMP to either enzyme. The catalytic reaction in both enzymes is restricted to the phosphorus atom and to the exocyclic oxygen atoms. Substitution of the negatively charged oxygen atom by an uncharged dimethylamino group in axial or equatorial position renders the compound non-hydrolyzable. Substitution of an exocyclic oxygen by a sulphur atom reduces the rate of the catalytic reaction about 100-fold if sulphur is placed in axial position and more than 10000-fold if sulphur is placed in equatorial position. A reaction mechanism for the enzymatic hydrolysis of cAMP is proposed.     

Dependence of thrombin- and trypsin-catalyzed hydrolysis of N-alpha-arylsulfonyl-L-arginine methyl esters on the structure of acylamide part of substrates]

For comparative studies on the esterase activities of thrombin and trypsin N(alpha)-arylsulfonyl-L-arginine methyl esters were synthetised containing in aromatic ring substituents of different polar nature, size and hydrophobicity. The kinetics of their hydrolysis by thrombin and trypsin were measured. Values of Km and kcat in steady-state conditions were determined. It was shown, that thrombin-catalysed hydrolysis was more sensitive than that of trypsin to the nature of substituents of arylsulfonyl group and determined by their polar and steric effects. A line correlation between specificity constants (kcat/Km) and sigma and Es of substituents were demonstrated. The difference in reactivity of compounds under investigation is suggested to depend on alterations of stability of hydrogen bond between arylsulfonylamide nitrogen atom of substrate and the active center of the enzyme due to changes in the acidity of the arylsulfonylamide group affected by substituent of the benzene ring.     

Influence of the separation of the charged groups and aromatic ring on interaction of tyrosine and phenylalanine analogues and derivatives with beta-cyclodextrin

Interactions of tyrosine and phenylalanine analogues with beta-cyclodextrin have been examined in terms of structural features of the ligand such as the separation of the charged amino group and aromatic ring, the presence of additional functional group attached to the amino or phenyl ring, and the presence of a charge on amino or carboxyl group, and steric effects using steady-state and time-resolved fluorescence spectroscopy and microcalorimetry. The studied aromatic amino acids possess low binding constant to beta-cyclodextrin, diversified with respect to the presence or absence of a substituent in para position of the phenyl ring. However, calculated, based on the global analysis of the fluorescence intensity decays, binding constants do not allow to estimate unequivocally the influence of the distance between the charged groups and phenol/phenyl ring on the inclusion complex stability because of their low diversification.     

Site-directed mutagenesis experiments on the putative deprotonation site of squalene-hopene cyclase from Alicyclobacillus acidocaldarius

To provide insight into the catalytic mechanism for the final deprotonation reaction of squalene-hopene cyclase (SHC) from Alicyclobacillus acidocaldarius, mutagenesis experiments were conducted for the following ten residues: Thr41, Glu45, Glu93, Arg127, Trp133, Gln262, Pro263, Tyr267, Phe434 and Phe437. An X-ray analysis of SHC has revealed that two types of water molecules ("front water" and "back waters") were involved around the deprotonation site. The results of these mutagenesis experiments allow us to propose the functions of these residues. The two residues of Gln262 and Pro263 probably work to keep away the isopropyl group of the hopanyl cation intermediate from the "front water molecule," that is, to place the "front water" in a favorable position, leading to the minimal production of by-products, i.e., hopanol and hop-21(22)-ene. The five residues of Thr41, Glu45, Glu93, Arg127 and Trp133, by which the hydrogen-bonded network incorporating the "back waters" is constructed, increase the polarization of the "front water" to facilitate proton elimination from the isopropyl moiety of the hopanyl cation, leading to the normal product, hop-22(29)-ene. The three aromatic residues of Tyr267, Phe434 and Phe437 are likely to play an important role in guiding squalene from the enzyme surface to the reaction cavity (substrate channeling) by the strong affinity of their aromatic residues to the squalene substrate.     

Control of substrate specificity by active-site residues in nitrobenzene dioxygenase

Nitrobenzene 1,2-dioxygenase from Comamonas sp. strain JS765 catalyzes the initial reaction in nitrobenzene degradation, forming catechol and nitrite. The enzyme also oxidizes the aromatic rings of mono- and dinitrotoluenes at the nitro-substituted carbon, but the basis for this specificity is not understood. In this study, site-directed mutagenesis was used to modify the active site of nitrobenzene dioxygenase, and the contribution of specific residues in controlling substrate specificity and enzyme performance was evaluated. The activities of six mutant enzymes indicated that the residues at positions 258, 293, and 350 in the alpha subunit are important for determining regiospecificity with nitroarene substrates and enantiospecificity with naphthalene. The results provide an explanation for the characteristic specificity with nitroarene substrates. Based on the structure of nitrobenzene dioxygenase, substitution of valine for the asparagine at position 258 should eliminate a hydrogen bond between the substrate nitro group and the amino group of asparagine. Up to 99% of the mononitrotoluene oxidation products formed by the N258V mutant were nitrobenzyl alcohols rather than catechols, supporting the importance of this hydrogen bond in positioning substrates in the active site for ring oxidation. Similar results were obtained with an I350F mutant, where the formation of the hydrogen bond appeared to be prevented by steric interference. The specificity of enzymes with substitutions at position 293 varied depending on the residue present. Compared to the wild type, the F293Q mutant was 2.5 times faster at oxidizing 2,6-dinitrotoluene while retaining a similar Km for the substrate based on product formation rates and whole-cell kinetics.     

Structural and mechanistic insights into homocysteine degradation by a mutant of methionine γ‐lyase based on substrate‐assisted catalysis

Methionine γ‐lyse (MGL) catalyzes the α, γ‐elimination of l ‐methionine and its derivatives as well as the α, β‐elimination of l ‐cysteine and its derivatives to produce α‐keto acids, volatile thiols, and ammonia. The reaction mechanism of MGL has been characterized by enzymological studies using several site‐directed mutants. The Pseudomonas putida MGL C116H mutant showed drastically reduced degradation activity toward methionine while retaining activity toward homocysteine. To understand the underlying mechanism and to discern the subtle differences between these substrates, we analyzed the crystal structures of the reaction intermediates. The complex formed between the C116H mutant and methionine demonstrated that a loop structure (Ala51–Asn64) in the adjacent subunit of the catalytic dimer cannot approach the cofactor pyridoxal 5′‐phosphate (PLP) because His116 disrupts the interaction of Asp241 with Lys240, and the liberated side chain of Lys240 causes steric hindrance with this loop. Conversely, in the complex formed between C116H mutant and homocysteine, the thiol moiety of the substrate conjugated with PLP offsets the imidazole ring of His116 via a water molecule, disrupting the interaction of His116 and Asp241 and restoring the interaction of Asp241 with Lys240. These structural data suggest that the Cys116 to His mutation renders the enzyme inactive toward the original substrate, but activity is restored when the substrate is homocysteine due to substrate‐assisted catalysis.     

Functional role of the "aromatic cage" in human monoamine oxidase B: structures and catalytic properties of Tyr435 mutant proteins

Current structural results of several flavin-dependent amine oxidizing enzymes including human monoamine oxidases A and B (MAO A and MAO B) show aromatic amino acid residues oriented approximately perpendicular to the flavin ring, suggesting a functional role in catalysis. In the case of human MAO B, two tyrosyl residues (Y398 and Y435) are found in the substrate binding site on the re face of the covalent flavin ring [Binda et al. (2002) J. Biol. Chem. 277, 23973-23976]. To probe the functional significance of this structure, Tyr435 in MAO B was mutated with the amino acids Phe, His, Leu, or Trp, the mutant proteins expressed in Pichia pastoris, and purified to homogeneity. Each mutant protein contains covalent FAD and exhibits a high level of catalytic functionality. No major alterations in active site structures are detected on comparison of their respective crystal structures with that of WT enzyme. The relative k(cat)/K(m) values for each mutant enzyme show Y435 > Y435F = Y435L = Y435H > Y435W. A similar behavior is also observed with the membrane-bound forms of MAO A and MAO B (MAO A Y444 mutant enzymes are found to be unstable on membrane extraction). p-Nitrobenzylamine is found to be a poor substrate while p-nitrophenethylamine is found to be a good substrate for all WT and mutant forms of MAO B. Analysis of these kinetic and structural data suggests the function of the "aromatic cage" in MAO to include a steric role in substrate binding and access to the flavin coenzyme and to increase the nucleophilicity of the substrate amine moiety. These results are consistent with a proposed polar nucleophilic mechanism for catalytic amine oxidation.     

Control of Substrate Specificity by Active-Site Residues in Nitrobenzene Dioxygenase

Nitrobenzene 1,2-dioxygenase from Comamonas sp. strain JS765 catalyzes the initial reaction in nitrobenzene degradation, forming catechol and nitrite. The enzyme also oxidizes the aromatic rings of mono- and dinitrotoluenes at the nitro-substituted carbon, but the basis for this specificity is not understood. In this study, site-directed mutagenesis was used to modify the active site of nitrobenzene dioxygenase, and the contribution of specific residues in controlling substrate specificity and enzyme performance was evaluated. The activities of six mutant enzymes indicated that the residues at positions 258, 293, and 350 in the α subunit are important for determining regiospecificity with nitroarene substrates and enantiospecificity with naphthalene. The results provide an explanation for the characteristic specificity with nitroarene substrates. Based on the structure of nitrobenzene dioxygenase, substitution of valine for the asparagine at position 258 should eliminate a hydrogen bond between the substrate nitro group and the amino group of asparagine. Up to 99% of the mononitrotoluene oxidation products formed by the N258V mutant were nitrobenzyl alcohols rather than catechols, supporting the importance of this hydrogen bond in positioning substrates in the active site for ring oxidation. Similar results were obtained with an I350F mutant, where the formation of the hydrogen bond appeared to be prevented by steric interference. The specificity of enzymes with substitutions at position 293 varied depending on the residue present. Compared to the wild type, the F293Q mutant was 2.5 times faster at oxidizing 2,6-dinitrotoluene while retaining a similar K_m for the substrate based on product formation rates and whole-cell kinetics.     

Anomeric specificity of D-xylose isomerase.

Crystal structures of complexes of D-xylose isomerase with deoxysugars have been determined. Deoxynojirimycin is a structural analogue of alpha-pyranose and mimics the binding of these aldose substrates. The structure of this complex supports the hypothesis that an imidazole group catalyzes ring opening of the pyranose. The steric restrictions in the active site of the enzyme prevent a beta-pyranose from binding in the same way. For the reverse reaction with ketoses, the anomeric specificity is less certain. Dideoxyimino-D-glucitol is a structural analogue of the ketose alpha-D-furanose. The binding of the inhibitor dideoxyimino-D-glucitol to the crystals of the enzyme does not mimic the binding of the reactive alpha-D-fructofuranose. Superposition of the nonphysiological substrate alpha-D-fructofuranose onto the atomic positions of dideoxyimino-D-glucitol is not possible due to the steric restrictions of the active site. However, by utilizing the approximate 2-fold symmetry of the sugar, a stereochemically sensible model is produced which is consistent with other data. In addition to reaction with alpha-D-furanose, the enzyme probably reacts with open ring keto sugars which are present at significant concentrations. Other sugars which resemble furanoses either do not inhibit significantly or are not observed in the crystals bound in a single conformation.     

Bacterial degradation of aromatic compounds via angular dioxygenation

Dioxygenation is one of the important initial reactions of the bacterial degradation of various aromatic compounds. Aromatic compounds, such as biphenyl, toluene, and naphthalene, are dioxygenated at lateral positions of the aromatic ring resulting in the formation of cis-dihydrodiol. This "normal" type of dioxygenation is termed lateral dioxygenation. On the other hand, the analysis of the bacterial degradation of fluorene (FN) analogues, such as 9-fluorenone, dibenzofuran (DF), carbazole (CAR), and dibenzothiophene (DBT)-sulfone, and DF-related diaryl ether compounds, dibenzo-p-dioxin (DD) and diphenyl ether (DE), revealed the presence of the novel mode of dioxygenation reaction for aromatic nucleus, generally termed angular dioxygenation. In this atypical dioxygenation, the carbon bonded to the carbonyl group in 9-fluorenone or to heteroatoms in the other compounds, and the adjacent carbon in the aromatic ring are both oxidized. Angular dioxygenation of DF, CAR, DBT-sulfone, DD, and DE produces the chemically unstable hemiacetal-like intermediates, which are spontaneously converted to 2,2',3-trihydroxybiphenyl, 2'-aminobiphenyl-2,3-diol, 2',3'-dihydroxybiphenyl-2-sulfinate, 2,2',3-trihydroxydiphenyl ether, and phenol and catechol, respectively. Thus, angular dioxygenation for these compounds results in the cleavage of the three-ring structure or DE structure. The angular dioxygenation product of 9-fluorenone, 1-hydro-1,1a-dihydroxy-9-fluorenone is a chemically stable cis-diol, and is enzymatically transformed to 2'-carboxy-2,3-dihydroxybiphenyl. 2'-Substituted 2,3-dihydroxybiphenyls formed by angular dioxygenation of FN analogues are degraded to monocyclic aromatic compounds by meta cleavage and hydrolysis. Thus, after the novel angular dioxygenation, subsequent degradation pathways are homologous to the corresponding part of that of biphenyl. Compared to the bacterial strains capable of catalyzing lateral dioxygenation, few bacteria having angular dioxygenase have been reported. Only a few degradation pathways, CAR-degradation pathway of Pseudomonas resinovorans strain CA10, DF/DD-degradation pathway of Sphingomonas wittichii strain RW1, DF/DD/FN-degradation pathway of Terrabacter sp. strain DBF63, and carboxylated DE-degradation pathway of P. pseudoalcaligenes strain POB310, have been investigated at the gene level. As a result of the phylogenetic analysis and the comparison of substrate specificity of angular dioxygenase, it is suggested that this atypical mode of dioxygenation is one of the oxygenation reactions originating from the relaxed substrate specificity of the Rieske nonheme iron oxygenase superfamily. Genetic characterization of the degradation pathways of these compounds suggests the possibility that the respective genetic elements constituting the entire catabolic pathway have been recruited from various other bacteria and/or other genetic loci, and that these pathways have not evolutionary matured.     

New EPSP synthase inhibitors: Synthesis and evaluation of an aromatic tetrahedral intermediate mimic containing a 3-malonate ether as a 3-phosphate surrogate

A new analog of the EPSP synthase enzyme reaction intermediate 1, containing a 3-malonate ether moiety in place of the usual 3-phosphate group, was synthesized from 3,5-dihydroxybenzoic acid. This simple, synthetically accessible aromatic compound (5) is an effective competitive inhibitor versus S3P with an apparent K₁ of 1.3 ± 0.22 μM. This result demonstrates that a simple benzene ring can be a suitable achiral substitute for the more complex shikimate ring in the design of EPSP synthase inhibitors. Furthermore, the greater potency of 5 versus the phenol 6, glycolate 7 and the gallic acid analog 8 demonstrates the requirement for multiple anionic charges at the dihydroxybenzoate 5-position in order to attain effective inhibition of this enzyme. However, this 3-malonate ether substituted compound was at least 10-fold less effective as a bisubstrate inhibitor than the corresponding 3-phosphate. This suggests that tetrahedral intermediate mimics possessing a 3-malonate ether moiety are less effective than their corresponding 3-phosphates in accessing the optimal enzyme conformation stabilizing 1.     

Sugar recognition by CscB and LacY

The sucrose permease (CscB) and lactose permease (LacY) of Escherichia coli belong to the oligosaccharide/H(+) symporter subfamily of the major facilitator superfamily, and both catalyze sugar/H(+) symport across the cytoplasmic membrane. Thus far, there is no common substrate for the two permeases; CscB transports sucrose, and LacY is highly specific for galactopyranosides. Determinants for CscB sugar specificity are unclear, but the structural organization of key residues involved in sugar binding appears to be similar in CscB and LacY. In this study, several sugars containing galactopyranosyl, glucopyranosyl, or fructofuranosyl moieties were tested for transport with cells overexpressing either CscB or LacY. CscB recognizes not only sucrose but also fructose and lactulose, but glucopyranosides are not transported and do not inhibit sucrose transport. The findings indicate that CscB exhibits practically no specificity with respect to the glucopyranosyl moiety of sucrose. Inhibition of sucrose transport by CscB tested with various fructofuranosides suggests that the C(3)-OH group of the fructofuranosyl ring may be important for recognition by CscB. Lactulose is readily transported by LacY, where specificity is directed toward the galactopyranosyl ring, and the affinity of LacY for lactulose is similar to that observed for lactose. The studies demonstrate that the substrate specificity of CscB is directed toward the fructofuranosyl moiety of the substrate, while the specificity of LacY is directed toward the galactopyranosyl moiety.     

Purification and characterization of a 1,2,4-trihydroxybenzene 1,2-dioxygenase from the basidiomycete Phanerochaete chrysosporium.

1,2,4-Trihydroxybenzene (THB) is an intermediate in the Phanerochaete chrysosporium degradation of vanillate and aromatic pollutants. A P. chrysosporium intracellular enzyme able to oxidatively cleave the aromatic ring of THB was purified by ammonium sulfate precipitation, hydrophobic and ion-exchange chromatographies, and native gel electrophoresis. The native protein has a molecular mass of 90 kDa and a subunit mass of 45 kDa. The enzyme catalyzes an intradiol cleavage of the substrate aromatic ring to produce maleylacetate. 18O2 incorporation studies demonstrate that molecular oxygen is a cosubstrate in the reaction. The enzyme exhibits high substrate specificity for THB; however, catechol cleavage occurs at approximately 20% of the optimal rate. THB dioxygenase catalyzes a key step in the degradation pathway of vanillate, an intermediate in lignin degradation. Maleylacetate, the product of THB cleavage, is reduced to beta-ketoadipate by an NADPH-requiring enzyme present in partially purified extracts.     

The catalytic mechanism of indole-3-glycerol phosphate synthase: crystal structures of complexes of the enzyme from Sulfolobus solfataricus with substrate analogue,substrate, and product

Indoleglycerol phosphate synthase catalyzes the ring closure of an N-alkylated anthranilate to a 3-alkyl indole derivative, a reaction requiring Lewis acid catalysis in vitro. Here, we investigated the enzymatic reaction mechanism through X-ray crystallography of complexes of the hyperthermostable enzyme from Sulfolobus solfataricus with the substrate 1-(o-carboxyphenylamino) 1-deoxyribulose 5-phosphate, a substrate analogue and the product indole-3-glycerol phosphate. The substrate and the substrate analogue are bound to the active site in a similar, extended conformation between the previously identified phosphate binding site and a hydrophobic pocket for the anthranilate moiety. This binding mode is unproductive, because the carbon atoms that are to be joined are too far apart. The indole ring of the bound product resides in a second hydrophobic pocket adjacent to that of the anthranilate moiety of the substrate. Although the hydrophobic moiety of the substrate moves during catalysis from one hydrophobic pocket to the other, the triosephosphate moiety remains rigidly bound to the same set of hydrogen-bonding residues. Simultaneously, the catalytically important residues Lys53, Lys110 and Glu159 maintain favourable distances to the atoms of the ligand undergoing covalent changes. On the basis of these data, the structures of two putative catalytic intermediates were modelled into the active site. This new structural information and the modelling studies provide further insight into the mechanism of enzyme-catalyzed indole synthesis. The charged epsilon-amino group of Lys110 is the general acid, and the carboxylate group of Glu159 is the general base. Lys53 guides the substrate undergoing conformational transitions during catalysis, by forming a salt-bridge to the carboxylate group of its anthranilate moiety.     

Studies on enzyme-substrate interactions of cholinephosphotransferase from rat liver

In order to elucidate the reaction mechanism and the substrate-binding sites, CDPcholine:1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2), prepared from rat liver microsomal fraction, has been subjected to kinetic analysis and substrate specificity studies. Kinetic evidence supports the hypothesis of a Bi-Bi sequential mechanism, involving a direct nucleophilic attack of diacylglycerol on CDPcholine during the reaction. To investigate the substrate requirements for recognition and catalysis, several CDPcholine analogs, modified in the nitrogen base or in the sugar or in the pyrophosphate bridge, have been synthesized, characterized and assayed as substrates and/or inhibitors of the reaction. The amino group on the pyrimidine ring, the 2'-alcoholic function of the ribose moiety as well as the pyrophosphate bridge have been identified as critical sites for enzyme-substrates interactions.     

Influence of substrate modification and C-terminal truncation on the active site structure of substrate-bound heme oxygenase from Neisseriae meningitidis. A 1H NMR study

Heme oxygenase (HO), from the pathogenic bacterium N. meningitidis(NmHO), which secures host iron, shares many properties with mammalian HOs but also exhibits some key differences. The crystal structure appears more compact, and the crystal-undetected C-terminus interacts with substrate in solution. The unique nature of substrate-protein, specifically pyrrole-I/II-helix-2, peripheral interactions in NmHO are probed by 2D (1)H NMR to reveal unique structural features controlling substrate orientation. The thermodynamics of substrate orientational isomerism are mapped for substrates with individual vinyl → methyl → hydrogen substitutions and with enzyme C-terminal deletions. NmHO exhibits significantly stronger orientational preference, reflecting much stronger and selective pyrrole-I/II interactions with the protein matrix, than in mammalian HOs. Thus, replacing bulky vinyls with hydrogens results in a 180° rotation of substrate about the α,γ-meso axis in the active site. A "collapse" of the substrate pocket as substrate size decreases is reflected in movement of helix-2 toward the substrate as indicated by significant and selective increased NOESY cross-peak intensity, increase in steric Fe-CN tilt reflected in the orientation of the major magnetic axis, and decrease in steric constraints controlling the rate of aromatic ring reorientation. The active site of NmHO appears "stressed" for native protohemin, and its "collapse" upon replacing vinyls by hydrogen leads to a factor ~10(2) increase in substrate affinity. Interaction of the C-terminus with the active site destabilizes the crystallographic protohemin orientation by ~0.7 kcal/mol, which is consistent with optimizing the His207-Asp27 H-bond. Implications of the active site "stress" for product release are discussed.     

Novel insights into the fungal oxidation of monoaromatic and biarylic environmental pollutants by characterization of two new ring cleavage enzymes

The phenol-degrading yeast Trichosporon mucoides can oxidize and detoxify biarylic environmental pollutants such as dibenzofuran, diphenyl ether and biphenyl by ring cleavage. The degradation pathways are well investigated, but the enzymes involved are not. The high similarity of hydroxylated biphenyl derivatives and phenol raised the question if the enzymes of the phenol degradation are involved in ring cleavage or whether specific enzymes are necessary. Purification of enzymes from T. mucoides with catechol cleavage activity demonstrated the existence of three different enzymes: a classical catechol-1,2-dioxygenase (CDO), not able to cleave the aromatic ring system of 3,4-dihydroxybiphenyl, and two novel enzymes with a high affinity towards 3,4-dihydroxybiphenyl. The comparison of the biochemical characteristics and mass spectrometric sequence data of these three enzymes demonstrated that they have different substrate specificities. CDO catalyzes the ortho-cleavage of dihydroxylated monoaromatic compounds, while the two novel enzymes carry out a similar reaction on biphenyl derivatives. The ring fission of 3,4-dihydroxybiphenyl by the purified enzymes results in the formation of (5-oxo-3-phenyl-2,5-dihydrofuran-2-yl)acetic acid. These results suggest that the ring cleavage enzymes catalyzing phenol degradation are not involved in the ring cleavage of biarylic compounds by this yeast, although some intermediates of the phenol metabolism may function as inducers.     

Discovery and role of methylidene imidazolone,a highly electrophilic prosthetic group

The elimination of ammonia from alpha-amino acids is a chemically difficult process. While the non-acidic beta-proton has to be abstracted, the much more acidic ammonium protons must remain untouched to maintain the leaving group ability of this positively charged group. Histidine and phenylalanine ammonia-lyases (HAL and PAL) possess a catalytically essential electrophilic group which has been believed to be dehydroalanine for 30 years. Recently, the X-ray structure of HAL has been solved. The electron density was not consistent with dehydroalanine but showed the presence of methylidene imidazolone (MIO) instead. The high electrophilicity of this prosthetic group as well as the geometry at the active site support a previously proposed mechanism involving a Friedel-Crafts-type attack at the aromatic ring of the substrate. Further biochemical evidence for this unprecedented electrophile-assisted ammonia elimination is also presented. Although no X-ray structure of PAL has been published as yet, spectrophotometrical evidence for the presence of MIO has been provided. Finally, a chemical model for the PAL reaction is described.