Active site residues of cis-2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase from Comamonas testosteroni strain B-356

cis-2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase (BphB) from Comamonas testosteroni strain B-356 is the second enzyme of the biphenyl/polychlorinated biphenyl degradation pathway. Based on the crystal structure of a related BphB, three conserved residues, Ser142, Tyr155, and Lys159, have been suggested to function as a "catalytic triad" as for other members of the short-chain alcohol dehydrogenase/reductase (SDR) family. In this study, substitution of each triad residue was examined in BphB. At pH 9.0, turnover numbers relative to wild-type enzyme were as follows: Y155F, 0.1%; S142A, 1%; and K159A, 10%. Although the Michaelis constants of K159A and S142A for cis-2,3-dihydro-2,3-dihydroxybiphenyl increased about 20-fold, relatively little change was observed in the K(m) for dinucleotide. The K159A mutant, which showed little dehydrogenase activity at pH 7, was sharply activated by increasing the pH, reaching almost 25% of the activity of the wild-type enzyme at pH 9. 8. These three residues are therefore critical for BphB activity, as suggested by the crystal structure and similarity to other SDR family members. In addition, BphB showed a strong preference for NAD(+) over NADP(+), with a 260-fold higher specificity constant (k(cat)/K(m)). Evidence is presented that the inefficient use of NADP(+) by BphB might partly be due to the presence of an aspartate residue at position 36.     

cis-2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase and cis-1, 2-dihydro-1,2-dihydroxynaphathalene dehydrogenase catalyze dehydrogenation of the same range of substrates.

Pseudomonas putida strain G7 cis-1,2-dihydro-1, 2-dihydroxynaphthalene dehydrogenase (NahB) and Comamonas testosteroni strain B-356 cis-2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase (BphB) were found to be catalytically active towards cis-2,3-dihydro-2,3-dihydroxybiphenyl (specificity factors of 501 and 5850 s-1 mM-1 respectively), cis-1,2-dihydro-1, 2-dihydroxynaphthalene (specificity factors of 204 and 193 s-1 mM-1 respectively) and 3,4-dihydro-3,4-dihydroxy-2,2',5, 5'-tetrachlorobiphenyl (specificity factors of 1.6 and 4.9 s-1 mM-1 respectively). A key finding in this work is the capacity of strain B-356 BphB as well as Burkholderia cepacia strain LB400 BphB to catalyze dehydrogenation of 3,4-dihydro-3,4-dihydroxy-2,2',5, 5'-tetrachlorobiphenyl which is the metabolite resulting from the catalytic meta-para hydroxylation of 2,2',5,5'-tetrachlorobiphenyl by LB400 biphenyl dioxygenase.     

Mechanistic studies on trans-2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase (Ent A) in the biosynthesis of the iron chelator enterobactin

The enzyme 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase (2,3-diDHB dehydrogenase, hereafter Ent A), the product of the enterobactin biosynthetic gene entA, catalyzes the NAD(+)-dependent oxidation of the dihydroaromatic substrate 2,3-dihydro-2,3-dihydroxybenzoate (2,3-diDHB) to the aromatic catecholic product 2,3-dihydroxybenzoate (2,3-DHB). The catechol 2,3-DHB is one of the key siderophore units of enterobactin, a potent iron chelator secreted by Escherichia coli. To probe the reaction mechanism of this oxidation, a variety of 2,3-diDHB analogues were synthesized and tested as substrates. Specifically, we set out to elucidate both the regio- and stereospecificity of alcohol oxidation as well as the stereochemistry of NAD+ reduction. Of those analogues tested, only those with a C3-hydroxyl group (but not a C2-hydroxyl group) were oxidized to the corresponding ketone products. Reversibility of the Ent A catalyzed reaction was demonstrated with the corresponding NADH-dependent reduction of 3-ketocyclohexane- and cyclohexene-1-carboxylates but not the 2-keto compounds. These results establish that Ent A functions as an alcohol dehydrogenase to specifically oxidize the C3-hydroxyl group of 2,3-diDHB to produce the corresponding 2-hydroxy-3-oxo-4,6-cyclohexadiene-1-carboxylate (Scheme II) as a transient species that undergoes rapid aromatization to give 2,3-DHB. Stereospecificity of the C3 allylic alcohol group oxidation was confirmed to be 3R in a 1R,3R dihydro substrate, 3, and hydride transfer occurs to the si face of enzyme-bound NAD+.     

A novel NAD-binding protein revealed by the crystal structure of 2,3-diketo-L-gulonate reductase (YiaK)

Escherichia coli YiaK catalyzes the reduction of 2,3-diketo-L-gulonate in the presence of NADH. It belongs to a large family of oxidoreductases that is conserved in archaea, bacteria, and eukaryotes but shows no sequence homology to other proteins. We report here the crystal structures at up to 2.0-A resolution of YiaK alone and in complex with NAD-tartrate. YiaK has a new polypeptide backbone fold and a novel mode of recognizing the NAD cofactor. In addition, NAD is bound in an unusual conformation, at the interface of a dimer of the enzyme. The crystallographic analysis unexpectedly revealed the binding of tartrate in the active site. Enzyme kinetics studies confirm that tartrate and the related D-malate are inhibitors of YiaK. In contrast to most other enzymes where substrate binding produces a more closed conformation, the binding of NAD-tartrate to YiaK produces a more open active site. The free enzyme conformation is incompatible with NAD binding. His(44) is likely the catalytic residue of the enzyme.     

Variation on a theme of SDR. dTDP-6-deoxy-L- lyxo-4-hexulose reductase (RmlD) shows a new Mg2+-dependent dimerization mode

dTDP-6-deoxy-L-lyxo-4-hexulose reductase (RmlD) catalyzes the final step in the conversion of dTDP-D-glucose to dTDP-L-rhamnose in an NAD(P)H- and Mg2+-dependent reaction. L-rhamnose biosynthesis is an antibacterial target. The structure of RmlD from Salmonella enterica serovar Typhimurium has been determined, and complexes with NADH, NADPH, and dTDP-L-rhamnose are reported. RmlD differs from other short chain dehydrogenases in that it has a novel dimer interface that contains Mg2+. Enzyme catalysis involves hydride transfer from the nicotinamide ring of the cofactor to the C4'-carbonyl group of the substrate. The substrate is activated through protonation by a conserved tyrosine. NAD(P)H is bound in a solvent-exposed cleft, allowing facile replacement. We suggest a novel role for the conserved serine/threonine residue of the catalytic triad of SDR enzymes.     

Crystal structure at 1.8 A resolution of CDP-D-glucose 4,6-dehydratase from Yersinia pseudotuberculosis

CDP-D-glucose 4,6-dehydratase catalyzes the conversion of CDP-D-glucose to CDP-4-keto-6-deoxyglucose in an NAD(+)-dependent manner. The product of this conversion is a building block for a variety of primary antigenic determinants in bacteria, possibly implicated directly in reactive arthritis. Here, we describe the solution of the high-resolution crystal structure of CDP-D-glucose 4,6-dehydratase from Yersinia pseudotuberculosis in the resting state. This structure represents the first CDP nucleotide utilizing dehydratase of the short-chain dehydrogenase/reductase (SDR) family to be determined, as well as the first tetrameric structure of the subfamily of SDR enzymes in which NAD(+) undergoes a full reaction cycle. On the basis of a comparison of this structure with structures of homologous enzymes, a chemical mechanism is proposed in which Tyr157 acts as the catalytic base, initiating hydride transfer by abstraction of the proton from the sugar 4'-hydroxyl. Concomitant with the removal of the proton from the 4'-hydroxyl oxygen, the sugar 4'-hydride is transferred to the B face of the NAD(+) cofactor, forming the reduced cofactor and a CDP-4-keto-d-glucose intermediate. A conserved Lys161 most likely acts to position the NAD(+) cofactor so that hydride transfer is favorable and/or to reduce the pK(a) of Tyr157. Following substrate oxidation, we propose that Lys134, acting as a base, would abstract the 5'-hydrogen of CDP-4-keto-D-glucose, priming the intermediate for the spontaneous loss of water. Finally, the resulting Delta(5,6)-glucoseen intermediate would be reduced suprafacially by the cofactor, and reprotonation at C-5' is likely mediated by Lys134.     

Steady-state kinetics and the inactivation by 2,3-butanedione of the energy-independent transhydrogenase of Escherichia coli cell membranes.

Kinetic measurements indicate that the energy-independent transhydrogenation of 3-acetylpyridine-NAD+ by NADPH in membranes of Escherichia coli follows a rapid equilibrium random bireactant mechanism. Each substrate, although reacting preferentially with its own binding site, is able to interact with the binding site of the other substrate to cause inhibition of enzyme activity. 5'-AMP (and ADP) and 2'-AMP interact with the NAD+- and NADP+-binding sites, respectively. Phenylglyoxal and 2,3-butanedione in borate buffer inhibit transhydrogenase activity presumably by reacting with arginyl residues. Protection against inhibition by 2,3-butanedione is afforded by NADP+, NAD+, and high concentrations of NADPH and NADH. Low concentrations of NADPH and NADH increase the rate of inhibition by 2,3-butanedione. Similar effects are observed for the inactivation of the transhydrogenase by tryptic digestion in the presence of these coenzymes. It is concluded that there are at least two conformations of the active site of the transhydrogenase which differ in the extent to which arginyl residues are accessible to exogenous agents such as trypsin and 2,3-butanedione. One conformation is induced by low concentrations of NADH and NADPH. Under these conditions the coenzymes could be reacting at the active site or at an allosteric site. The stimulation of transhydrogenase activity by low concentrations of the NADH is consistent with the latter possibility.     

Determinants of the dual cofactor specificity and substrate cooperativity of the human mitochondrial NAD(P)+-dependent malic enzyme: functional roles of glutamine 362

The human mitochondrial NAD(P)+-dependent malic enzyme (m-NAD-ME) is a malic enzyme isoform with dual cofactor specificity and substrate binding cooperativity. Previous kinetic studies have suggested that Lys362 in the pigeon cytosolic NADP+-dependent malic enzyme has remarkable effects on the binding of NADP+ to the enzyme and on the catalytic power of the enzyme (Kuo, C. C., Tsai, L. C., Chin, T. Y., Chang, G.-G., and Chou, W. Y. (2000) Biochem. Biophys. Res. Commun. 270, 821-825). In this study, we investigate the important role of Gln362 in the transformation of cofactor specificity from NAD+ to NADP+ in human m-NAD-ME. Our kinetic data clearly indicate that the Q362K mutant shifted its cofactor preference from NAD+ to NADP+. The Km(NADP) and kcat(NADP) values for this mutant were reduced by 4-6-fold and increased by 5-10-fold, respectively, compared with those for the wild-type enzyme. Furthermore, up to a 2-fold reduction in Km(NADP)/Km(NAD) and elevation of kcat(NADP)/kcat(NAD) were observed for the Q362K enzyme. Mutation of Gln362 to Ala or Asn did not shift its cofactor preference. The Km(NADP)/Km(NAD) and kcat(NADP)/kcat(NAD) values for Q362A and Q362N were comparable with those for the wild-type enzyme. The DeltaG values for Q362A and Q362N with either NAD+ or NADP+ were positive, indicating that substitution of Gln with Ala or Asn at position 362 brings about unfavorable cofactor binding at the active site and thus significantly reduces the catalytic efficiency. Our data also indicate that the cooperative binding of malate became insignificant in human m-NAD-ME upon mutation of Gln362 to Lys because the sigmoidal phenomenon appearing in the wild-type enzyme was much less obvious that that in Q362K. Therefore, mutation of Gln362 to Lys in human m-NAD-ME alters its kinetic properties of cofactor preference, malate binding cooperativity, and allosteric regulation by fumarate. However, the other Gln362 mutants, Q362A and Q362N, have conserved malate binding cooperativity and NAD+ specificity. In this study, we provide clear evidence that the single mutation of Gln362 to Lys in human m-NAD-ME changes it to an NADP+-dependent enzyme, which is characteristic because it is non-allosteric, non-cooperative, and NADP+-specific.     

Crystal structure and mechanism of tryptophan 2,3-dioxygenase, a heme enzyme involved in tryptophan catabolism and in quinolinate biosynthesis

The structure of tryptophan 2,3-dioxygenase (TDO) from Ralstonia metallidurans was determined at 2.4 A. TDO catalyzes the irreversible oxidation of l-tryptophan to N-formyl kynurenine, which is the initial step in tryptophan catabolism. TDO is a heme-containing enzyme and is highly specific for its substrate l-tryptophan. The structure is a tetramer with a heme cofactor bound at each active site. The monomeric fold, as well as the heme binding site, is similar to that of the large domain of indoleamine 2,3-dioxygenase, an enzyme that catalyzes the same reaction except with a broader substrate tolerance. Modeling of the putative (S)-tryptophan hydroperoxide intermediate into the active site, as well as substrate analogue and mutagenesis studies, are consistent with a Criegee mechanism for the reaction.     

Characterization and functional role of Saccharomyces cerevisiae 2,3-butanediol dehydrogenase

Using a conserved sequence motif, a new gene (YAL060W) of the MDR family has been identified in Saccharomyces cerevisiae. The expressed protein was a stereoespecific (2R,3R)-2,3-butanediol dehydrogenase (BDH). The best substrates were (2R,3R)-2,3-butanediol for the oxidation and (3R/3S)-acetoin and 1-hydroxy-2-propanone for the reduction reactions. The enzyme is extremely specific for NAD(H) as cofactor, probably because the presence of Glu223 in the cofactor binding site, instead of the highly conserved Asp223. BDH is inhibited competitively by 4-methylpyrazole with a K(i) of 34 microM. Yeast could grow on 2,3-butanediol or acetoin as a sole energy and carbon sources, and a 3.6-fold increase in BDH activity was observed when cells were grown in 2,3-butanediol, suggesting a role of the enzyme in 2,3-butanediol metabolism. However, the disruption of the YAL060W gene was not lethal for the yeast under laboratory conditions, and the disrupted strain could also grow in 2,3-butanediol and acetoin. This suggests that other enzymes, in addition to BDH, can also metabolize 2,3-butanediol in yeast.     

Crystal structure of meso-2,3-butanediol dehydrogenase in a complex with NAD+ and inhibitor mercaptoethanol at 1.7 A resolution for understanding of chiral substrate recognition mechanisms

The crystal structure of a ternary complex of meso-2,3-butanediol dehydrogenase with NAD+ and a competitive inhibitor, mercaptoethanol, has been determined at 1.7 A resolution by means of molecular replacement and refined to a final R-factor of 0.194. The overall structure is similar to those of the other short chain dehydrogenase/reductase enzymes. The NAD+ binding site, and the positions of catalytic residues Ser139, Tyr152, and Lys156 are also conserved. The crystal structure revealed that mercaptoethanol bound specifically to meso-2,3-butanediol dehydrogenase. Two residues around the active site, Gln140 and Gly183, forming hydrogen bonds with the inhibitor, are important but not sufficient for distinguishing stereoisomerism of a chiral substrate.     

Temperature-dependent structure of the E x S complex of Bacillus stearothermophilus alcohol dehydrogenase

The catalytic chemistry of the thermophilic Bacillus stearothermophilus alcohol dehydrogenase (HtADH) closely resembles that of mesophilic horse liver alcohol dehydrogenase (HLADH). Molecular dynamics (MD) simulations of the htADH x NAD+ x EtO- complex at 298, 323, and 348 K show that the structure of the ligated Zn2+...EtO- complex varies slightly with change in temperature. The MD-created Boltzmann distribution of htADH x NAD+ x EtO- structures establishes the formation of multiple states which increase in number with a decrease in temperature. The motions of the cofactor domain are highly correlated with the motions of NAD+ at the optimal growth temperature (348 K), with NAD+ being pushed toward the substrate by Val260. With a decrease in temperature, the motion together of the cofactor and substrate is reversed, and at 298 K, the nicotinamide ring of the cofactor moves away from the substrate. Both the distance between and the angle of approach of C4 of NAD+ and HD of EtO- become distorted from those of the reactive conformation. The percentages of ground state present as the reactive conformation at different temperatures are approximately correlated with the kcat for the htADH enzymatic reaction. The rate constant for the htADH x NAD+ x EtOH --> htADH x NAD+ x EtO- proton dissociation, which is mediated by Thr40-OH, becomes slower at lower temperatures. The time-dependent distance between EtO- and Thr40-OH reveals that the Thr40 hydroxyl group sways between the substrate and NAD+ ribose 2'-hydroxyl group at the optimal enzyme growth temperature, and this movement is effectively frozen out as the temperature decreases. The temperature dependence of active site conformations is due to the change in both long-range and short-range motions of the E x S complex.     

Characterization and functional role of Saccharomyces cerevisiae 2,3-butanediol dehydrogenase

Using a conserved sequence motif, a new gene (YAL060W) of the MDR family has been identified in Saccharomyces cerevisiae. The expressed protein was a stereoespecific (2R,3R)-2,3-butanediol dehydrogenase (BDH). The best substrates were (2R,3R)-2,3-butanediol for the oxidation and (3R/3S)-acetoin and 1-hydroxy-2-propanone for the reduction reactions. The enzyme is extremely specific for NAD(H) as cofactor, probably because the presence of Glu223 in the cofactor binding site, instead of the highly conserved Asp223. BDH is inhibited competitively by 4-methylpyrazole with a K_i of 34 μM. Yeast could grow on 2,3-butanediol or acetoin as a sole energy and carbon sources, and a 3.6-fold increase in BDH activity was observed when cells were grown in 2,3-butanediol, suggesting a role of the enzyme in 2,3-butanediol metabolism. However, the disruption of the YAL060W gene was not lethal for the yeast under laboratory conditions, and the disrupted strain could also grow in 2,3-butanediol and acetoin. This suggests that other enzymes, in addition to BDH, can also metabolize 2,3-butanediol in yeast.     

Inhibition of pyridine-nucleotide-dependent enzymes by pyrazoles. Synthesis and enzymology of a novel A-ring pyrazole steroid

A novel A-ring pyrazole steroid, 2,3-bisaza-A-nor-1,5(10)-estradien-17 beta-ol (3), was synthesized as a potential inhibitor of steroidal NAD(P)H-dependent oxidoreductases. Compound 3 proved to be a potent inhibitor of 3(17)beta-hydroxysteroid dehydrogenase (from P. testosteroni) exhibiting a Ki of 90 +/- 20 nM. The activities of 3 alpha,20 beta-hydroxysteroid dehydrogenase (from S. hydrogenans), steroid-5 alpha-reductase (from rat prostate), and 3 alpha-hydroxysteroid dehydrogenase (from rat liver) were unaffected by pyrazole 3. Dead end inhibition studies indicate an ordered binding of cofactor prior to substrate or pyrazole inhibitor.     

Dithiol- and NAD-dependent degradation of epoxyalkanes by Xanthobacter Py2

A broad range of epoxyalkanes was converted into the corresponding ketones by cell extracts of Xanthobacter Py2. Both 1,2- and 2,3-epoxyalkanes were degraded and in addition, the degradation of 2,3-epoxyalkanes in all cases was highly enantioselective. Conversion of a deuterium-labelled substrate indicated that the ketone product was probably formed indirectly via an hydroxy intermediate. Degradation of epoxyalkanes by Xanthobacter Py2 was dependent on both NAD and another, not yet identified, cofactor that was present in the low-molecular-mass fraction (LMF) of propene-grown cells. It is proposed that the LMF was involved in a reductive reaction step since it could be replaced by dithiothreitol (DTT) and various other dithiol compounds. Epoxyalkane-degrading activity was inhibited by the sulphhydryl blocking reagent N-ethylmaleimide (NEM). Inhibition by NEM and stimulation by LMF, DTT and other dithiols was effective only in the simultaneous presence of NAD.     

Structural rearrangement accompanying NAD+ synthesis within a bacterial DNA ligase crystal

DNA ligase is an enzyme important for DNA repair and replication. Eukaryotic genomes encode ligases requiring ATP as the cofactor; bacterial genomes encode NAD(+)-dependent ligase. This difference in substrate specificities and the essentiality of NAD(+)-dependent ligase for bacterial survival make NAD(+)-dependent ligase a good target for designing highly specific anti-infectives. Any such structure-guided effort would require the knowledge of the precise mechanism of NAD+ recognition by the enzyme. We report the principles of NAD+ recognition by presenting the synthesis of NAD+ from nicotinamide mononucleotide (NMN) and AMP, catalyzed by Enterococcus faecalis ligase within the crystal lattice. Unprecedented conformational change, required to reorient the two subdomains of the protein for the condensation to occur and to recognize NAD+, is captured in two structures obtained using the same protein crystal. Structural data and sequence analysis presented here confirms and extends prior functional studies of the ligase adenylation reaction.     

A kinetic locking-on strategy for bioaffinity purification: further studies with alcohol dehydrogenase

The kinetic locking-on strategy improves the selectivity of protein purification procedures based on immobilized cofactor derivatives through use of enzyme-specific substrate analogues in irrigants to promote biospecific adsorption. This paper describes the development and application of this strategy to the one-chromatographic step affinity purification of NAD(P)+-dependent alcohol dehydrogenases using 8'-azo-linked immobilized NAD(P)+, S6-linked and N6-linked immobilized NAD+, and N6-linked immobilized NADP+ derivatives. These studies were carried out using alcohol dehydrogenases from Saccharomyces cerevisiae (YADH, EC 1.1.1.1), equine liver (HLADH, EC 1.1.1.1), and Thermoanaerobium brockii (TBADH, EC 1.1.1.2). The results reveal that the factors which require careful consideration before development of a truly biospecific system based on the locking-on strategy include: (i) the stability of the immobilized cofactor derivative; (ii) the spacer-arm composition of the affinity derivative; (iii) the accessible immobilized cofactor concentration; (iv) the soluble locking-on ligand concentration; (v) the dissociation constant of locking-on ligand, and (vi) the identification and elimination of nonbiospecific interference. The S6-linked immobilized NAD+ derivative (synthesized with a hydrophilic spacer arm) proved to be the most suitable of the affinity adsorbents investigated in the present study for use with the locking-on strategy. This conclusion was based primarily on the observations that this affinity adsorbent was stable, retained cofactor activity with the "test" enzymes under study, and was not prone to nonbiospecific interactions. Using this immobilized derivative in conjunction with the locking-on strategy, alcohol dehydrogenase from Saccharomyces cerevisiae was purified to electrophoretic homogeneity in a single affinity chromatographic step.     

Characterization of active recombinant 2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase from Comamonas testosteroni B-356 and sequence of the encoding gene (bphB).

2,3-Dihydro-2,3-dihydroxybiphenyl-2,3-dehydrogenase (B2,3D) catalyzes the second step in the biphenyl degradation pathway. The nucleotide sequence of Comamonas testosteroni B-356 bphB, which encodes B2,3D, was determined. Structural analysis showed that the dehydrogenases involved in the bacterial degradation of aromatic compounds are related to each other and that their phylogenetic relationships are very similar to the relationships observed for dioxygenases that catalyze the initial reaction in the degradation pathway. The bphB sequence was used to produce recombinant active His-tagged B2,3D, which allowed us to describe for the first time some of the main features of a B2,3D. This enzyme requires NAD+, its optimal pH is 9.5, and its native M(r) was found to be 123,000, which makes it a tetramer. These characteristics are very similar to those reported for the related enzyme cis-toluene dihydrodiol dehydrogenase. The Km value and maximum rate of metabolism for 2,3-dihydro-2,3-dihydroxybiphenyl were 73 +/- 16 microM and 46 +/- 4 nmol min-1 microgram-1, respectively. Compared with the cis-toluene dihydrodiol dehydrogenase, B2,3D appeared to be more substrate specific since it was unable to attack cis-1,2-dihydroxy-cyclohexa-3,5-diene.     

Biochemical and structural characterization of a short-chain dehydrogenase/reductase of Thermus thermophilus HB8: a hyperthermostable aldose-1-dehydrogenase with broad substrate specificity

Thermus thermophilus HB8 is a hyperthermophilic bacterium, thriving at environmental temperature near 80 degrees C. The genomic analysis of this bacterium predicted 18 genes for proteins belonging to the short-chain dehydrogenase/reductases (SDR) superfamily, but their functions remain unknown. A SDR encoded in a gene (TTHA0369) was chosen for functional and structural characterization. Enzymatic assays revealed that the recombinant tetrameric protein has a catalytic activity as NAD(+)-dependent aldose 1-dehydroganse, which accepts various aldoses such as d-fucose, d-galactose, d-glucose, l-arabinose, cellobiose and lactose. The enzyme also oxidized non-sugar alicyclic alcohols, and was competitively inhibited by hexestrol, 1,10-phenanthroline, 2,3-benzofuran and indole. The enzyme was stable at pH 2-13 and up to 85 degrees C. We have determined the crystal structure of the enzyme-NAD(+) binary complex at 1.65A resolution. The structure provided evidence for the strict coenzyme specificity and broad substrate specificity of the enzyme. Additionally, it has unusual features, aromatic-aromatic interactions among Phe141 and Phe249 in the subunit interface and hydrogen networks around the C-terminal Asp-Gly-Gly sequence at positions 242-244. Stability analysis of the mutant D242N, F141A and F249A enzymes indicated that the two unique structural features contribute to the hyperthermostability of the enzyme. This study demonstrates that aldose 1-dehydrogenase is a member of the SDR superfamily, and provides a novel structural basis of thermostability.     

Dihydroxylation and dechlorination of chlorinated biphenyls by purified biphenyl 2,3-dioxygenase from Pseudomonas sp. strain LB400.

Oxidation of biphenyl and nine chlorinated biphenyls (CBs) by the biphenyl 2,3-dioxygenase from Pseudomonas sp. strain LB400 was examined. The purified terminal oxygenase required the addition of partially purified electron transport components, NAD(P)H, and ferrous iron to oxidize biphenyl and CBs. cis-Biphenyl 2,3-dihydrodiol was produced with biphenyl as the substrate. Dihydrodiols were produced from all CBs, and more than one compound was produced with most substrates. Catechols were produced when the dioxygenase-catalyzed reaction occurred at the 2,3 position of a 2-chlorophenyl ring, resulting in dechlorination of the substrate. Oxidation at the 3,4 position of a 2,5-dichlorophenyl ring produced a 3,4-dihydrodiol. Compounds resulting from both types of reaction were produced during oxidation of 2,5,2'-trichlorobiphenyl. The broad substrate specificity and the ability to oxidize at different ring positions suggest that the biphenyl 2,3-dioxygenase is responsible for the wide range of CBs oxidized by Pseudomonas sp. strain LB400.