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The mechanism by which molecular oxygen is activated by the organic cofactor pyridoxal phosphate (PLP) for oxidation reactions remains poorly understood. Recent work has identified arginine oxidases that catalyze desaturation or hydroxylation reactions. Here, we investigate a desaturase from the Pseudoalteromonas luteoviolacea indolmycin pathway. Our work, combining X-ray crystallographic, biochemical, spectroscopic, and computational studies, supports a shared mechanism with arginine hydroxylases, involving two rounds of single-electron transfer to oxygen and superoxide rebound at the 4' carbon of the PLP cofactor. The precise positioning of a water molecule in the active site is proposed to control the final reaction outcome. This proposed mechanism provides a unified framework to understand how oxygen can be activated by PLP-dependent enzymes for oxidation of arginine and elucidates a shared mechanistic pathway and intertwined evolutionary history for arginine desaturases and hydroxylases.

CouR, a MarR-type transcriptional repressor, regulates the cou genes, encoding p-hydroxycinnamate catabolism in the soil bacterium Rhodococcus jostii RHA1. The CouR dimer bound two molecules of the catabolite p-coumaroyl-CoA (Kd = 11 ± 1 μM). The presence of p-coumaroyl-CoA, but neither p-coumarate nor CoASH, abrogated CouR's binding to its operator DNA in vitro. The crystal structures of ligand-free CouR and its p-coumaroyl-CoA-bound form showed no significant conformational differences, in contrast to other MarR regulators. The CouR-p-coumaroyl-CoA structure revealed two ligand molecules bound to the CouR dimer with their phenolic moieties occupying equivalent hydrophobic pockets in each protomer and their CoA moieties adopting non-equivalent positions to mask the regulator's predicted DNA-binding surface. More specifically, the CoA phosphates formed salt bridges with predicted DNA-binding residues Arg36 and Arg38, changing the overall charge of the DNA-binding surface. The substitution of either arginine with alanine completely abrogated the ability of CouR to bind DNA. By contrast, the R36A/R38A double variant retained a relatively high affinity for p-coumaroyl-CoA (Kd = 89 ± 6 μM). Together, our data point to a novel mechanism of action in which the ligand abrogates the repressor's ability to bind DNA by steric occlusion of key DNA-binding residues and charge repulsion of the DNA backbone.

The 80-kb dit cluster of Burkhoideria xenovorans LB400 encodes the catabolism of abietane diterpenoids. This cluster includes ditQ and ditU, predicted to encode cytochromes P450 (P450s) belonging to the poorly characterized CYP226A subfamily. Using proteomics, we identified 16 dit-encoded proteins that were significantly more abundant in LB400 cells grown on dehydroabietic acid (DhA) or abietic acid (AbA) than in succinategrown cells. A key difference in the catabolism of DhA and AbA lies in the differential expression of the P450s; DitU was detected only in the AbA-grown cells, whereas DitQ was expressed both during growth on DhA and during growth on AbA. Analyses of insertion mutants showed that ditQ was required for growth on DhA, ditU was required for growth on AbA, and neither gene was required for growth on the central intermediate, 7-oxo-DhA. In cell suspension assays, patterns of substrate removal and metabolite accumulation confirmed the role of DitU in AbA transformation and the role of DitQ in DhA transformation. Spectral assays revealed that DitQ binds both DhA (dissociation constant, 0.98 [+ or -] 0.01 [micro]M) and palustric acid. Finally, DitQ transformed DhA to 7-hydroxy-DhA in vitro. These results demonstrate the distinct roles of the P450s DitQ and DitU in the transformation of DhA and AbA, respectively, to 7-oxo-DhA in a convergent degradation pathway.

Proteomics and targeted gene disruption were used to investigate the catabolism of benzene, styrene, biphenyl, and ethylbenzene in Rhodococcusjostii RHAI, a well-studied soil bacterium whose potent polychlorinated biphenyl (PCB)-transforming properties are partly due to the presence of the related Bph and Etb pathways. Of 151 identified proteins, 22 Bph/Etb proteins were among the most abundant in biphenyl-, ethylbenzene-, benzene-, and styrene-grown cells. Cells grown on biphenyl, ethylbenzene, or benzene contained both Bph and Etb enzymes and at least two sets of lower Bph pathway enzymes. By contrast, styrene-grown cells contained no Etb enzymes and only one set of lower Bph pathway enzymes. Gene disruption established that biphenyl dioxygenase (BPDO) was essential for growth of RHA1 on benzene or styrene but that ethylbenzene dioxygenase (EBDO) was not required for growth on any of the tested substrates. Moreover, whole-cell assays of the [DELTA]bphAa and etbAal::cmrA etbAa2::aphH mutants demonstrated that while both dioxygenases preferentially transformed biphenyl, only BPDO transformed styrene. Deletion of pcaL of the [beta]-ketoadipate pathway disrupted growth on benzene but not other substrates. Thus, styrene and benzene are degraded via meta-and ortho-cleavage, respectively. Finally, catalases were more abundant during growth on nonpolar aromatic compounds than on aromatic acids. This suggests that the relaxed specificities of BPDO and EBDO that enable RHA1 to grow on a range of compounds come at the cost of increased uncoupling during the latter's initial transformation. The stress response may augment RHA1's ability to degrade PCBs and other pollutants that induce similar uncoupling.

Biphenyl dioxygenase (BPDO) catalyzes the aerobic transformation of biphenyl and various polychlorinated biphenyls (PCBs). In three different assays, [BPDO.sub.B356] from Pandoraea pnomenusa B-356 was a more potent PCB-degrading enzyme than [BPDO.sub.LB400] from Burkholderia xenovorans LB400 (75% amino acid sequence identity), transforming nine congeners in the following order of preference: 2,3',4-trichloro ~2,3,4'-trichloro > 3,3'-dichloro > 2,4,4'-trichloro > 4,4'-dichloro ~2,2'-dichloro > 2,6-dichioro > 2~2',3,3'-tetrachloro ~2,2',5,5'-tetrachloro. Except for 2,2',5,5'-tetrachlorobiphenyl, [BPDO.sub.B356] transformed each congener at a higher rate than [BPDO.sub.LB400]. The assays used either whole cells or purified enzymes and either individual congeners or mixtures of congeners. Product analyses established previously unrecognized [BPDO.sub.B356] activities, including the 3,4-dihydroxylation of 2,6-dichlorobiphenyl. [BPDO.sub.LB400] had a greater apparent specificity for biphenyl than [BPDO.sub.B356] ([k.sub.cat]/[K.sub.m] = 2.4 x [10.sup.6] [+ or -] 0.7 x [10.sup.6] [M.sup.-1] [s.sup.-1] versus [k.sub.cat]/[K.sub.m] = 0.21 x [10.sup.6] [+ or -] 0.04 x [10.sup.6] [M.sup.-1] [s.sup.-1]). However, the latter transformed biphenyl at a higher maximal rate ([k.sub.cat] = 4.1 [+ or -] 0.2 [s.sup.-1] versus [k.sub.cat] = 0.4 [+ or -] 0.1 [s.sup.-1]). A variant of [BPDO.sub.LB400] containing four active site residues of [BPDO.sub.B356] transformed para-substituted congeners better than [BPDO.sub.LB400]. Interestingly, a substitution remote from the active site, A267S, increased the enzyme's preference for meta-substituted congeners. Moreover, this substitution had a greater effect on the kinetics of biphenyl utilization than substitutions in the substrate-binding pocket. In all variants, the degree of coupling between congener depletion and [O.sub.2] consumption was approximately proportional to congener depletion. At 2.4-[Angstrom] resolution, the crystal structure of the [BPDO.sub.B356]-2,6-dichlorobiphenyl complex, the first crystal structure of a BPDO-PCB complex, provided additional insight into the reactivity of this isozyme with this congener, as well as into the differences in congener preferences of the BPDOs.

Sphingomonas wittichii RW1 degrades chlorinated dibenzofurans and dibenzo-p-dioxins via meta cleavage. We used inverse PCR to amplify dxnB2, a gene encoding one of three meta-cleavage product (MCP) hydrolases identified in the organism that are homologues of BphD involved in biphenyl catabolism. Purified DxnB2 catalyzed the hydrolysis of 8-OH 2-hydroxy-6-oxo-6- phenylhexa-2,4-dienoate (HOPDA) approximately six times faster than for HOPDA at saturating substrate concentrations. Moreover, the specificity of DxnB2 for HOPDA ([k.sub.cat]/[K.sub.m] = 1.2 x [10.sup.7] [M.sup.-1] [s.sup.-1]) was about half that of the BphDs of Burkholderia xenovorans LB400 and Rhodococcus globerulus P6, two potent polychlorinated biphenyl (PCB)-degrading strains. Interestingly, DxnB2 transformed 3-Cl and 4-OH HOPDAs, compounds that inhibit the BphDs and limit PCB degradation. DxnB2 had a higher specificity for 9-Cl HOPDA than for HOPDA but a lower specificity for 8-CI HOPDA ([k.sub.cat]/[K.sub.m]= 1.7 x 106 [M.sup.-1] [s.sup.-1]), the chlorinated analog of 8-OH HOPDA produced during dibenzofuran catabolism. Phylogenetic analyses based on structure-guided sequence alignment revealed that DxnB2 belongs to a previously unrecognized class of MCP hydrolases, evolutionarily divergent from the BphDs although the physiological substrates of both enzyme types are HOPDAs. However, both classes of enzymes have mainly small hydrophobic residues lining the subsite that binds the [C.sup.-]6 phenyl of HOPDA, in contrast to the bulky hydrophobic residues (Phel06, Phe135, Trpl50, and Phe197) found in the class II enzymes that prefer substrates possessing a [C.sup.-]6 alkyl. Thr196 and/or Asn203 appears to be an important determinant of specificity for DxnB2, potentially forming hydrogen bonds with the 8-OH substituent. This study demonstrates that the substrate specificities of evolutionarily divergent hydrolases may be useful for degrading mixtures of pollutants, such as PCBs.

Phthalate isomers and their esters are important pollutants whose biodegradation is not well understood. Rhodococcus sp. strain RHA1 is notable for its ability to degrade a wide range of aromatic compounds. RHA1 was previously shown to degrade phthalate (PTH) and to have genes putatively encoding terephthalate (TPA) degradation. Transcriptomic analysis of 8,213 genes indicated that 150 were up-regulated during growth on PTH and that 521 were up-regulated during growth on TPA. Distinct ring cleavage dioxygenase systems were differentially expressed during growth on PTH and TPA. Genes encoding the protocatechuate (PCA) pathway were induced on both substrates, while genes encoding the catechol branch of the PCA pathway were additionally induced only on TPA. Accordingly, protocatechuate-3,4-dioxygenase activity was induced in cells grown on both substrates, while catechol-1,2-dioxygenase activity was induced only in cells grown on TPA. Knockout analysis indicated that pcaL, encoding 3-oxoadipate enol-lactone hydrolase and 4-carboxymuconolactone decarboxylase, was required for growth on both substrates but that pcaB, encoding [BETA]-carboxy-cis,cis-muconate lactonizing enzyme, was required for growth on PTH only. These results indicate that PTH is degraded solely via the PCA pathway, whereas TPA is degraded via a bifurcated pathway that additionally includes the catechol branch of the PCA pathway.

The complete genome sequence of Rhodococcus sp. strain RHA1 is analyzed in the biphenyl pathway (BPH) and a microarray with probe for 8,313 genes is developed and employed. The analysis suggests that up to 22 enzymes, including 8 newly identified ones, may function in the BPH pathway of RHA1.

BphK is a glutathione S-transferase of unclear physiological function that occurs in some bacterial biphenyl catabolic (bph) pathways. We demonstrated that BphK of Burkholderia xenovorans strain LB400 catalyzes the dehalogenation of 3-chloro 2-hydroxy-6-oxo-6-phenyl-2,4-dienoates (HOPDAs), compounds that are produced by the cometabolism of polychlorinated biphenyls (PCBs) by the bph pathway and that inhibit the pathway's hydrolase. A one-column protocol was developed to purify heterologously produced BphK. The purified enzyme had the greatest specificity for 3-Cl HOPDA ([k.sub.cat]/[K.sub.m], ~[10.sup.4] [M.sup.-1] [s.sup.-1]), which it dechlorinated approximately 3 orders of magnitude more efficiently than 4-chlorobenzoate, a previously proposed substrate of BphK. The enzyme also catalyzed the dechlorination of 5-Cl: HOPDA and 3,9,11-triCl HOPDA. By contrast, BphK did not detectably transform HOPDA, 4-Cl HOPDA, or chlorinated 2,3-dihydroxybiphenyls. The BphK-catalyzed dehalogenation proceeded via a ternary-complex mechanism and consumed 2 equivalents of glutathione (GSH) ([K.sub.m] for GSH in the presence of 3-Cl HOPDA, ~0.1 mM). A reaction mechanism consistent with the enzyme's specificity is proposed. The ability of BphK to dehalogenate inhibitory PCB metabolites supports the hypothesis that this enzyme was recruited to facilitate PCB degradation by the bph pathway.

In gram-negative bacteria, a pathway for aerobic degradation of phenylacetic acid (PAA) that proceeds via phenylacetyl-coenzyme A (COA) and hydrolytic ring fission plays a central role in the degradation of a range of aromatic compounds. In contrast, the PAA pathway and its role are not well characterized in gram-positive bacteria. A cluster including 13paa genes encoding enzymes orthologous to those of gram-negative bacteria was identified on the chromosome ofRhodococcus sp. strain RHAI. These genes were transcribed during growth on PAA, with 11 of the genes apparently in an operon yielding a single transcript. Quantitative proteomic analyses revealed that at least 146 proteins were more than twice as abundant in PAA-grown cells of RHAI than in pyruvate-grown cells. Of these proteins, 29 were identified, including 8 encoded by the paa genes. Knockout mutagenesis indicated that paaN, encoding a putative ring-opening enzyme, was essential for growth on PAA. However, paaF, encoding phenylacetyl-CoA ligase, and paaR, encoding a putative regulator, were not essential. paaN was also essential for growth of RHAI on phenylacetaldehyde, phenylpyruvate, 4-phenylbutyrate, 2-phenylethanol, 2-phenylethylamine, and L-phenylalanine. In contrast, growth on 3-hydroxyphenylacetate, ethylbenzene, and styrene was unaffected. These results suggest that the range of substrates degraded via the PAA pathway in RHAI is somewhat limited relative to the range in previously characterized gram-negative bacteria.

Genomic and proteomic approaches were used to investigate phthalate and benzoate catabolism in Rhodococcus sp. strain RHA1, a polychlorinated biphenyl-degrading actinomycete. Sequence analyses identified genes involved in the catabolism of benzoate (ben) and phthalate (pad), the uptake of phthalate (put), and two branches of the [beta]-ketoadipate pathway (catRABC and pcaJIHGBLFR). The regulatory and structural ben genes are separated by genes encoding a cytochrome P450. The pad and pat genes are contained on a catabolic island that is duplicated on plasmids pRHL1 and pRHL2 and includes predicted terephthalate catabolic genes (tpa). Proteomic analyses demonstrated that the [beta]-ketoadipate pathway is functionally convergent. Specifically, the pad and pat gene products were only detected in phthalate-grown cells. Similarly, the ben and cat gene products were only detected in benzoate-grown cells. However, pca-encoded enzymes were present under both growth conditions. Activity assays for key enzymes confirmed these results. Disruption of pcaL, which encodes a fusion enzyme, abolished growth on phthalate. In contrast, after a lag phase, growth of the mutant on benzoate was similar to that of the wild type. Proteomic analyses revealed 20 proteins in the mutant that were not detected in wild-type cells during growth on benzoate, including a CatD homolog that apparently compensated for loss of PcaL. Analysis of completed bacterial genomes indicates that the convergent [beta]-ketoadipate pathway and some aspects of its genetic organization are characteristic of rhodococci and related actinomycetes. In contrast, the high redundancy of catabolic pathways and enzymes appears to be unique to RHA1 and may increase its potential to adapt to new carbon sources.

The reactivities of four evolutionarily divergent extradiol dioxygenases towards mono-, di-, and trichlorihated (triCl) 2,3-dihydroxybiphenyls (DHBs) were investigated: 2,3-dihydroxybiphenyl dioxygenase (EC 1.13.11.39) from Burkholderia sp. strain LB400 (DHB[D.sub.LB400]), DHB[D.sub.P6]-I and DHB[D.sub.P6]-III from Rhodococcus globerulus P6, and 2,2',3-trihydroxybiphenyl dioxygenase from Sphingomonas sp. strain RW1 (THB[D.sub.RW1]). The specificity of each isozyme for particular DHBs differed by up to 3 orders of magnitude. Interestingly, the [K.sup.app.m] values of each isozyme for the tested polychlorinated DHBs were invariably lower than those of monochlorihated DHBs. Moreover, each enzyme cleaved at least one of the tested chlorinated (Cl) DHBs better than it cleaved DHB (e.g., apparent specificity constants for 3',5'-dichlorinated [diCl] DHB were 2 to 13.4 times higher than for DHB). These results are consistent with structural data and modeling studies which indicate that the substrate-binding pocket of the DHBDs is hydrophohic and can accommodate the Cl DHBs, particularly in the distal portion of the pocket. Although the activity of DHB[D.sub.P6]-III was generally lower than that of the other three enzymes, six of eight tested Cl DHBs were better substrates for DHB[D.sub.P6]-III than was DHB. Indeed, DHB[D.sub.P6]-III had the highest apparent specificity for 4,3',5'-triCl DHB and cleaved this compound better than two of the other enzymes. Of the four enzymes, THB[D.sub.RW1] had the highest specificity for 2'-Cl DHB and was at least five times more resistant to inactivation by 2'-Cl DHB, consistent with the similarity between the latter and 2,2',3-trihydroxybiphenyl. Nonetheless, THB[D.sub.RW1] had the lowest specificity for 2',6'-diCl DHB and, like the other enzymes, was unable to cleave this critical PCB metabolite (k.sup.app.sub.cat] < 0.001 [s.sup.-1]).

Rhodococcus sp. strain RHA1, a potent polychlorinated-biphenyl (PCB)-degrading strain, contains three linear plasmids ranging in size from 330 to 1,100 kb. As part of a genome sequencing project, we report here the complete sequence and characterization of the smallest and least-well-characterized of the RHA1 plasmids, pRHL3. The plasmid is an actinomycete invertron, containing large terminal inverted repeats with a tightly associated protein and a predicted open reading frame (ORF) that is similar to that of a mycobacterial rep gene. The pRHL3 plasmid has 300 putative genes, almost 21% of which are predicted to have a catabolic function. Most of these are organized into three clusters. One of the catabolic clusters was predicted to include limonene degradation genes. Consistent with this prediction, RHA1 grew on limonene, carveol, or carvone as the sole carbon source. The plasmid carries three cytochrome P450-encoding (CYP) genes, a finding consistent with the high number of CYP genes found in other actinomycetes. Two of the CYP genes appear to belong to novel families; the third belongs to CYP family 116 but appears to belong to a novel class based on the predicted domain structure of its reductase. Analyses indicate that pRHL3 also contains four putative 'genomic islands' (likely to have been acquired by horizontal transfer), insertion sequence elements, 19 transposase genes, and a duplication that spans two ORFs. One of the genomic islands appears to encode resistance to heavy metals. The plasmid does not appear to contain any housekeeping genes. However, each of the three catabolic clusters contains related genes that appear to be involved in glucose metabolism.

Toluate dioxygenase of Pseudomonas putida mt-2 ([TADO.sub.mt2]) and benzoate dioxygenase of Acinetobacter calcoaceticus ADP1 ([BADO.sub.ADP1]) catalyze the 1,2-dihydroxylation of different ranges of benzoates. The catalytic component of these enzymes is an oxygenase consisting of two subunits. To investigate the structural determinants of substrate specificity in these ring-hydroxylating dioxygenases, hybrid oxygenases consisting of the subunit of one enzyme and the [beta] subunit of the other were prepared, and their respective specificities were compared to those of the parent enzymes. Reconstituted [BADO.sub.ADP1] utilized four of the seven tested benzoates in the following order of apparent specificity: benzoate > 3-methylbenzoate > 3-chlorobenzoate > 2-methylbenzoate. This is a significantly narrower apparent specificity than for [TADO.sub.mt2] (3-methylbenzoate > benzoate ~ 3-chlorobenzoate > 4-methylbenzoate ~ 4-chlorobenzoate [much greater than] 2-methylbenzoate ~ 2-chlorobenzoate [Y. Ge, F. H. Vaillancourt, N. Y. Agar, and L. D. Eltis, J. Bacteriol. 184:4096-4103, 2002]). The apparent substrate specificity of the [[alpha].sub.b][[beta].sub.T] hybrid oxygenase for these benzoates corresponded to that of [BADO.sub.ADP1], the parent from which the [alpha] subunit originated. In contrast, the apparent substrate specificity of the [[alpha].sub.T][[beta].sub.B] hybrid oxygenase differed slightly from that of [TADO.sub.mt2] > (3-chlorobenzoate > 3-methyibienzoate > benzoate ~ 4-methylbenzoate > 4-chlorobenzoate > 2-methylbenzoate > 2-chiorobenzoate). Moreover, the [[alpha].sub.T][[beta].sub.B] hybrid catalyzed the 1,6-dihyiroxyliation of 2-methyilbenzoate, not the 1,2-dihydroxylation catalyzed by the [TADO.sub.mt2] parent. Finally, the turnover of this ortho-substituted benzoate was much better coupled to [O.sub.2] utilization in the hybrid than in the parent. Overall, these results support the notion that the [alpha] subunit harbors the principal determinants of specificity in ring-hydroxylating dioxygenases. However, they also demonstrate that the [beta] subunit contributes significantly to the enzyme's function.

Pseudomonas putida toluate dioxygenase activity with the effector XylS is influenced by both meta- and ortho-substituted benzoates, reflecting vestigial activity of the effector in regulating the TOL meta operon. Data suggest that the specificity of XylS reflects a dual regulatory role.

Research demonstrates that significant diversity exists in the aerobic transformation of polychlorinated biphenyls by divergent microorganisms mediating the degradation of biphenyls as revealed by Burkholderia and Rhodococcus species. Data indicate that hydrolase is a key determinant in the chemical transformation process.

The reduction potential of the recombinant BphF is 157 plus or minus 2 mV with the EPR spectrum of the reduced recombinant BphF resembling typical Rieske cluster. The protein readily oligomerizes in solution with no influence on the crystal's reduction potential and crystals reveal a head-to-tail interaction.

A study was conducted to examine the capability of the dehydrogenase and ring cleavage dioxygenase of the naphthalene degradation pathway to change 3,4-dihydroxylated biphenyl metabolites. Escherichia coli M15(pREP4) and SG13009 were utilized as bacterial strains while protein characterization was carried out using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results indicated that the recruitment of catabolic enzymes supporting specificities supporting enzymes of the biphenyl degradation pathway may lead to the degradation of polychlorinated biphenyls.

The magnitude of the stabilization of the heme-protein complex caused by the formation of hydrogen bonds by heme propionate groups of both myoglobin (Mb) and cytochrome b5 with adjacent surface amino acid residues was investigated. The kinetics of dissociation from variants of horse heart Mb and cytochrome b5 in which these hydrogen bonding interactions have been eliminated were studied. Findings showed that the interactions between the heme propionates and adjacent protein residues have a partial contribution to the stabilization of the heme-protein complex and are a major contributor in the kinetic trapping of the minor heme orientation.

An investigation of the evolutionary relationships among extradiol dioxygenases revealed only nine strictly conserved residues in the alignment of 35 extradiol dioxygenases. Six of these are the three metal ligands, His-146, His-210 and Glu-260 in BphC_LB400, and three additional active site residues, His-195, His-241 and Tyr-250. A number of structurally important residues that are remote from the active site were also detected. Phylogenetic analyses based on this alignment suggest that the ancestral extradiol dioxygenase was a one-domain enzyme and that the two-domain enzymes came from a single genetic duplication occurrence.

The dbfB gene of the enzyme 2,2', 3-trihydroxybiphenyl dioxygenase, a monomeric protein, is cloned from a cosmid library of the Sphingomonas strain which degrades dibenzofuran and dibenzo-p-dioxin. Kinetic evaluation of the enzyme yields similar K(m) values for 2,3-dihydroxybiphenyl and 2,2',-3-trihydroxybiphenyl.

The complete sequence of a 182-kb plasmid, pGKT2, from Gordonia sp. KTR9 is described. The studies have shown that hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) degradation by KTR9 is integrated with central nitrogen metabolism and that the uptake of RDX by bacterial cells does not need a dedicated transporter.

Microarrays are used for studying the mineralization of the most common oxysterol, 7-ketocholesterol (7KC), Rhodococcus jostii RHA1. The growth on 7KC versus growth on cholesterol has resulted in many differentially expressed genes, including the upregulation of two large gene clusters putatively encoding steroid catabolism.

X-ray crystallography and steady-state kinetics were used to study the interaction of copper-containing dissimilatory nitrite reductase from Alcaligenes faecalis S-6 (AfNiR) with each of the five small molecules. The results provide insight into the significant role of Asp98 and Ile257 mutants in inhibition mechanism of enzyme AfNiR.

The effects of the propane on the gene expression and biological removal of the water contaminant N-nitrosodimethylamine (NDMA) are examined. The results have shown that propane monooxygenase is responsible for the degradation of NDMA by Rhodococcus sp. strain RHA1 and have explained the increased cometabolic degradation of NDMA in the presence of propane.

The catalytic mechanism of [(BphD).sub.LB400] was investigated using steady-state and transient kinetic analyses together with structural analyses of the wild-type enzyme and a complex prepared by incubation of the latter with 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA). The study provides the first full spectra of catalytic intermediates for a meta-cleavage product (MCP) hydrolase and reveals key enzyme-substrate interactions, which allow to predict the catalytic roles of active site residues.

The use of protein-film voltammetry (PFV) is described to compare the redox behavior of three different Rieske proteins, RsRp, TtRp and BphF. The importance of the coupling between oxidation state and histidine protonation state is used to determine reduction potentials of Rieske clusters.

Research has been conducted on chlorinated 2,3-dixydroxybiphenyls. The physiological significance of these evolutionary divergent dixydroxybiphenyls in a single bacterium has been investigated and discussed.