Purification and characterization of 2'aminobiphenyl-2,3-diol 1,2-dioxygenase from Pseudomonas sp. LD2

Carbazole is a nitrogen-containing heteroaromatic compound that occurs as a widespread and mutagenic environmental pollutant. The 2'aminobiphenyl-2,3-diol 1,2-dioxygenase involved in carbazole degradation was purified to near electrophoretic homogeneity from Pseudomonas sp. LD2 by a combination of ion-exchange chromatography, ammonium sulfate precipitation, and hydrophobic interaction chromatography. This purification was challenging due to the great instability of the enzyme under many standard conditions. The enzyme was also purified to electrophoretic homogeneity from recombinant Escherichia coli expressing the 2'aminobiphenyl-2,3-diol 1,2-dioxygenase-encoding gene cloned from Pseudomonas sp. LD2. The molecular mass of the native enzyme was determined by gel filtration to be 70 kDa. The subunit molecular masses were determined to be 25 and 8 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the dioxygenase is an [alpha2beta2] heterotetramer. The optimal temperature and pH for the enzymatic production of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) from 2,3-dihydroxybiphenyl were determined to be 40 degrees C and 8.0, respectively. The maximum observed specific activity on 2,3-dihydroxybiphenyl was 48.1 mmol HOPDA min(-1) mg(-1). This indicated a maximum observed turnover rate of 360,000 molecules HOPDA enz(-1) s(-1). The K'm inhibition constant Ks and Vmax on 2,3 dihydroxybiphenyl were determined to be 5 microM, 37 microM, and 44 mmol min(-1) mg(-1), respectively. These results show that 2'aminobiphenyl-2,3-diol 1,2-dioxygenase is a meta-cleavage enzyme related to the 4,5-protocatechuate dioxygenase family, with comparable purification challenges posed by intrinsic enzyme instability.     

Cloning and nucleotide sequence of carbazole catabolic genes from Pseudomonas stutzeri strain OM1, isolated from activated sludge

A new carbazole (CAR)-degrading bacterium, called strain OM1, was isolated from activated sludge obtained from sewage disposal plants in Fukuoka Prefecture, and it was identified as Pseudomonas stutzeri. Anthranilic acid (AN), 2'-aminobiphenyl-2,3-diol and its meta-cleavage product, 2-hydroxy-6-oxo-6-(2'-aminophenyl)-hexa-2,4-dienoic acid, were identified as metabolic intermediates of CAR in the ethyl acetate extract of the culture broth. Therefore, the CAR catabolic pathway to AN in strain OM1 was indicated to be identical to those found in the Pseudomonas sp. strains CA06 and CA10. The strain OM1 degraded catechol (CAT) via a meta-cleavage pathway in contrast to strains CA06 and CA10, which transform catechol into cis, cis-munonic acid. Clones containing a 6.9-kb EcoRI fragment and a 3-kb PstI-SphI fragment were isolated from colonies, forming a clear zone of CAR and a yellow ring-cleavage product from CAT, respectively. Recombinant E. coli carrying the 6.9-kb fragment degraded CAR in the L-broth and produced AN. Cell-free extract from the clone carrying a 3-kb PstI-SphI fragment had high meta-ring-cleavage dioxygenase activity for CAT. The nucleotide sequences of these fragments were determined. The 6.9-kb fragment showed a very high degree of homology with the CAR catabolic genes of strain CA10. The amino acid and nucleotide sequences of the 3-kb fragment were found to exhibit significant homology with the genes for the CAT-catabolic enzymes of TOL plasmid pWW0, plasmid NAH7, and plasmid pVI150.     

Characterization of a sialidase (neuraminidase) isolated from Clostridium chauvoei (Jakari strain)

A sialidase from Clostridium chauvoei (Jakari strain), an indigenous bacterial strain that causes blackleg in Nigerian cattle and other ruminants was isolated and partially purified by chromatography on DEAE cellulose, hydroxyapatite and phenyl agarose columns. The enzyme migrated as a 65-kDa protein after electrophoresis on sodium dodecyl sulphate polyacrylamide gels. It was optimally active at pH 4.5 and 40 degrees C with an activation energy (Ea) of 13.40 kJ mol(-1). It had Km and Vmax values of 170 microM and 200 micromole h(-1) mg(-1) respectively with fetuin as substrate. When sialyllactose (Neu5Ac2,3 lactose) was used as substrate the Km and Vmax values were 8 microM and 5 micromoles min(-1) mg(-1) respectively. The Clostridium chauvoei sialidase cleaved sialic acids from RBC ghosts of sheep, horse, goat, cattle, pig and mice as well as mouse brain cells, albeit at different rates. The enzyme was activated by Ca2+ and Mg2+ and inhibited by the group-specific reagents diethylpyrocarbonate (DEP) and N-ethylmalemide (NEM). The sialidase inhibitors, 2,3 didehydroneuraminic acid (Neu5Ac2,3en) and paranitrophenyl oxamic acid (pNPO) inhibited the enzyme competitively with Ki values of 40 and 30 microM respectively.     

A novel meta-cleavage dioxygenase that cleaves a carboxyl-group-substituted 2-aminophenol. Purification and characterization of 4-amino-3-hydroxybenzoate 2,3-dioxygenase from Bordetella sp. strain 10d.

A bacterial strain that grew on 4-amino-3-hydroxybenzoic acid was isolated from farm soil. The isolate, strain 10d, was identified as a species of Bordetella. Cell extracts of Bordetella sp. strain 10d grown on 4-amino-3-hydroxybenzoic acid contained an enzyme that cleaved this substrate. The enzyme was purified to homogeneity with a 110-fold increase in specific activity. The purified enzyme was characterized as a meta-cleavage dioxygenase that catalyzed the ring fission between C2 and C3 of 4-amino-3-hydroxybenzoic acid, with the consumption of 1 mol of O2 per mol of substrate. The enzyme was therefore designated as 4-amino-3-hydroxybenzoate 2,3-dioxygenase. The molecular mass of the native enzyme was 40 kDa based on gel filtration; the enzyme is composed of two identical 21-kDa subunits according to SDS/PAGE. The enzyme showed a high dioxygenase activity only for 4-amino-3-hydroxybenzoic acid. The Km and Vmax values for this substrate were 35 micro m and 12 micro mol.min-1.(mg protein)-1, respectively. Of the 2-aminophenols tested, only 4-aminoresorcinol and 6-amino-m-cresol inhibited the enzyme. The enzyme reported here differs from previously reported extradiol dioxygenases, including 2-aminophenol 1,6-dioxygenase, in molecular mass, subunit structure and catalytic properties.     

Expression and homology modeling of 2′-aminobiphenyl-2,3-diol-1,2-dioxygenase from Pseudomonas stutzeri carbazole degradation pathway

The enzyme 2′-aminobiphenyl-2,3-diol-1,2-dioxygenase (CarB), encoded by two genes (carBa and carBb), is an α₂β₂ heterotetramer that presents meta-cleavage activity toward the hydroxylated aromatic ring in the carbazole degradation pathway from petroleum-degrader bacteria Pseudomonas spp. The 1082-base, pair polymerase chain reaction product corresponding to, carBaBb genes from Pseudomonas stutzeri ATCC 31258 was cloned by site-specific recombination and expressed in high levels in Escherichia coli BL21-SI with a histidine-tag and in native form. The CarB activity toward 2,3-dihydroxybiphenyl was similar for these two constructions. The α₂β₂ 3D model of CarB dioxygenase was proposed by homology modeling using the protocatechuate 4,5-dioxygenase (LigAB) structure as template. Accordingly, His12, His53, and Glu230 coordinate the Fe(II) in the catalytic site at the subunit CarBb. The model also indicates that His182 is the catalytic base responsible for deprotonating one of the hydroxyl group of the substrate by a hydrogen bond. The hydrophobic residues Trp257 and Phe258 in the CarB structure substituted the LigAB amino acid residues Ser269 and Asn270. These data could explain why the CarB was active for 2,3-dihydroxybiphenyl and not for protocatechuate.     

Isolation and characterization of Sphingomonas sp. GTIN11 capable of carbazole metabolism in petroleum

A bacterial culture was isolated from a manufactured gas plant (MGP) soil based on its ability to metabolize the nitrogen-containing heterocycle carbazole. The culture was identified as a Sphingomonas sp. and was given the designation GTIN11. A cloned 4.2kb DNA fragment was confirmed to contain genes responsible for carbazole degradation. DNA sequence analysis revealed that the fragment contained five open reading frames (ORFs) with the deduced amino acid sequence showing homology to; carbazole terminal dioxygenase (ORF1), 2,3-dihydroxybiphenyl dioxygenase subunits (ORF2 and ORF3), meta-cleavage compound hydrolases (ORF4), and ferrodoxin component of bacterial multicomponent dioxygenases (ORF5). The percent similarity was 61% of these proteins or less to known proteins. The specific activity of Sphingomonas sp. GTIN11 for the degradation of carbazole at 37 degrees C was determined to be 8.0 micromol carbazole degraded/min/g dry cell. This strain is unique in expressing the carbazole degradation trait constitutively. Resting cells of Sphingomonas sp. GTIN11 removed 95% of carbazole and 50% of C1-carbazoles from petroleum in a 16-h treatment time.     

Purification and characterization of catechol 2,3-dioxygenase from the aniline degradation pathway of Acinetobacter sp. YAA and its mutant enzyme, which resists substrate inhibition

Catechol 2,3-dioxygenase (C23O), a key enzyme in the meta-cleavage pathway of catechol metabolism, was purified from cell extract of recombinant Escherichia coli JM109 harboring the C23O gene (atdB) cloned from an aniline-degrading bacterium Acinetobacter sp. YAA. SDS-polyacrylamide gel electrophoresis and gel filtration chromatography analysis suggested that the enzyme (AtdB) has a molecular mass of 35 kDa as a monomer and forms a tetrameric structure. It showed relative meta-cleavage activities for the following catechols tested: catechol (100%), 3-methylcatechol (19%), 4-methylcatechol (57%), 4-chlorocatechol (46%), and 2,3-dihydroxybiphenyl (5%). To elevate the activity, a DNA self-shuffling experiment was carried out using the atdB gene. One mutant enzyme, named AtdBE286K, was obtained. It had one amino acid substitution, E286K, and showed 2.4-fold higher C23O activity than the wild-type enzyme at 100 microM. Kinetic analysis of these enzymes revealed that the wild-type enzyme suffered from substrate inhibition at >2 microM, while the mutant enzyme loosened substrate inhibition.     

Purification and characterization of dihydrofolate reductase from wild-type and trimethoprim-resistant Mycobacterium smegmatis

Dihydrofolate reductase (DHFR) from extracts of Mycobacterium smegmatis strain mc2(6) and trimethoprim-resistant mutant mc2(26) was purified to homogeneity. In crude extracts, the specific activity of the enzyme from the trimethoprim resistant strain was comparable to that from the sensitive strain. The DHFR from both sources was purified using affinity chromatography on MTX-Sepharose followed by Mono Q FPLC. The enzyme has an apparent molecular mass of 23 kDa from gel filtration on Sephadex G-100 and from SDS-PAGE. Amino terminal sequence analysis showed homology with DHFRs from a subset of other gram-positive organisms. The purified enzyme from the trimethoprim-sensitive organism exhibited Km values for H2folate and NADPH of 0.68 +/- 0.2 microM and 21 +/- 4 microM, respectively. The Km values for H2folate and NADPH for the enzyme from the drug-resistant organism were 1.8 +/- 0.4 microM and 5.3 +/- 1.5 microM, respectively. A kcat of 4.5 sec-1 was determined for the DHFR from both sources. The enzyme from both sources was competitively inhibited by pyrimethamine and trimethoprim. The Ki value of trimethoprim, for the enzyme from the drug-resistant organism was about six-fold higher than for the enzyme from drug-sensitive strain. Our data suggest that mutation of DHFR contributes to trimethoprim resistance in the mc2(26) strain of M. smegmatis.     

Kinetic properties of 5-carboxymethyl-2-hydroxymuconate semialdehyde dehydrogenase from Escherichia coli

Kinetic properties of purified 5-carboxymethyl-2-hydroxymuconate semialdehyde (CHMSA) dehydrogenase (EC 1.2.1.-) in the 4-hydroxyphenylacetate meta-cleavage pathway from Escherichia coli have been studied. The temperature--activity relationship for the enzyme from 27 to 45 degrees C showed an Arrhenius plot with an inflexion at 36 degrees C. When 5-carboxymethyl-2-hydroxymuconic semialdehyde and NAD were used as variable substrates, the double reciprocal plots were all linear and the lines intersected at one point below the horizontal axis, suggesting that a sequential mechanism is operating. From the replots of intercepts and slopes against reciprocal substrate concentrations were calculated Km (CHMSA) = 9.0 +/- 1.02 microM, Km (NAD) = 29.1 +/- 4.65 microM and the value for the dissociation constant of enzyme--NAD complex = 6.3 +/- 1.21 microM. ATP and the product of the reaction (NADH) acted as competitive inhibitors of the enzyme with respect to NAD. Apparent Ki values, estimated from Dixon plots, were 25.0 +/- 3.5 and 88.0 +/- 22.1 microM for NADH and ATP, respectively.     

Cloning and expression of genes encoding meta-cleavage enzymes from 4,6-dimethyldibenzothiophene-degrading Sphingomonas strain TZS-7

Sphingomonas strain TZS-7 was reported as the first strain to have the ability to degrade 4,6-dimethyldibenzothiophene (4,6-dmDBT) by the ring-destructive pathway. Two genes for meta-cleavage dioxygenases were cloned from strain TZS-7. Expression of each gene showed that one enzyme was specific for 2,3-dihydroxybiphenyl while another was more specific for catechol. The genes for the two enzymes were named dmdC and catA. The analysis of deduced amino acid sequences indicates that CatA falls into the class of meta-cleavage dioxygenases acting on dihydroxylated monocyclic compounds and DmdC falls into the class of meta-cleavage dioxygenases acting on dihydroxylated polycyclic compounds.     

Metabolism of 2,2'-dihydroxybiphenyl by Pseudomonas sp. strain HBP1: production and consumption of 2,2',3-trihydroxybiphenyl.

Cells of Pseudomonas sp. strain HBP1 grown on 2-hydroxy- or 2,2'-dihydroxybiphenyl contain NADH-dependent monooxygenase activity that hydroxylates 2,2'-dihydroxybiphenyl. The product of this reaction was identified as 2,2',3-trihydroxybiphenyl by 1H nuclear magnetic resonance and mass spectrometry. Furthermore, the monooxygenase activity also hydroxylates 2,2',3-trihydroxybiphenyl at the C-3' position, yielding 2,2',3,3'-tetrahydroxybiphenyl as a product. An estradiol ring cleavage dioxygenase activity that acts on both 2,2',3-tri- and 2,2',3,3'-tetrahydroxybiphenyl was partially purified. Both substrates yielded yellow meta-cleavage compounds that were identified as 2-hydroxy-6-(2-hydroxyphenyl)-6-oxo-2,4-hexadienoic acid and 2-hydroxy-6-(2,3-dihydroxyphenyl)-6-oxo-2,4-hexadienoic acid, respectively, by gas chromatography-mass spectrometry analysis of their respective trimethylsilyl derivatives. The meta-cleavage products were not stable in aqueous incubation mixtures but gave rise to their cyclization products, 3-(chroman-4-on-2-yl)pyruvate and 3-(8-hydroxychroman-4-on-2-yl)pyruvate, respectively. In contrast to the meta-cleavage compounds, which were turned over to salicylic acid and 2,3-dihydroxybenzoic acid, the cyclization products are not substrates to the meta-cleavage product hydrolase activity. NADH-dependent salicylate monooxygenase activity catalyzed the conversions of salicylic acid and 2,3-dihydroxybenzoic acid to catechol and pyrogallol, respectively. The partially purified estradiol ring cleavage dioxygenase activity that acted on the hydroxybiphenyls also produced 2-hydroxymuconic semialdehyde and 2-hydroxymuconic acid from catechol and pyrogallol, respectively.     

Degradation of chlorinated biphenyl,dibenzofuran, and dibenzo-p-dioxin by marine bacteria that degrade biphenyl,carbazole, or dibenzofuran

Marine bacterial strains (BP-PH, CAR-SF, and DBF-MAK) were isolated using biphenyl, carbazole (CAR), or dibenzofuran (DF) respectively as substrates for growth. Their 16S ribosomal DNA sequences showed that the species closest to strain BP-PH, strain CAR-SF, and strain DBF-MAK are Alteromonas macleodii (96.3% identity), Neptunomonas naphthovorans (93.1% identity), and Cycloclasticus pugetii (97.3% identity), respectively. The metabolites produced suggested that strain CAR-SF degrades CAR via dioxygenation in the angular position and by the meta-cleavage pathway, and that strain DBF-MAK degrades DF via both lateral and angular dioxygenation. Polychlorinated biphenyl (KC-300) and 2,3-dichlorodibenzo-p-dioxin were partially degraded by strain BP-PH and strain DBF-MAK, while 2,7-dichlorodibenzo-p-dioxin and 2,4,8-trichlorodibenzofuran remained virtually unchanged.     

Construction of a 3-chlorobiphenyl-utilizing recombinant from an intergeneric mating.

Recombinant Pseudomonas sp. strain CB15, which grows on 3-chlorobiphenyl (3CB), was constructed from Pseudomonas sp. strain HF1, which grows on 3-chlorobenzoate, and from Acinetobacter sp. strain P6, which grows on biphenyl, by using a continuous amalgamated culture apparatus. DNA from strains CB15 and HF1 hybridized very strongly to each other, while hybridization between both parental strains, HF1 and P6, was negligible. However, DNA from the recombinant CB15 hybridized moderately to strongly with three specific fragments of parental strain P6. Strains HF1 and P6 did not grow on 3CB, but recombinant strain CB15 mineralized this compound and released inorganic chloride. When growing on 3CB, strain CB15 accumulated brown products, one of which was identified as 3-chloro-5-(2'-hydroxy-3'-chlorophenyl)-1,2-benzoquinone by mass spectrometry. Emulsification and mechanical fragmentation greatly increased the rate of 3CB mineralization by strain CB15. At least three methods of inhibition from catecholic intermediates may account for slow growth on 3CB. The meta fission of 2,3-dihydroxybiphenyl (the nonchlorinated analog of the metabolic intermediate 3-chloro-2',3'-dihydroxybiphenyl) was affected by substrate inhibition (Vmax = 359 nmol.min-1.mg-1, Km = 114 microM, Kss [the inhibition constant] = 951 microM) and was also inhibited by 3-chlorocatechol. The ortho fission of 3-chlorocatechol, a degradation product, followed Michaelis-Menten kinetics (Vmax = 365 nmol.min-1.mg-1, Km = 1 microM), but the addition of 2,3-dihydroxybiphenyl inhibited the reaction (Ki = 0.87 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Isozymes of phosphoglyceromutase from the developing endosperm of Ricinus communis: isolation and kinetic properties

The isozymes of phosphoglyceromutase from the developing endosperm of Ricinus communis have been partially purified. The purified cytosolic and plastid isozymes have specific activities of 622.8 and 83.8 mumol min-1 mg protein-1, respectively. They both have relative molecular masses of approximately 64,000. The cytosolic enzyme has lower Km values for both 2-phosphoglycerate and 3-phosphoglycerate than the plastid enzyme. The Km values for 3-phosphoglycerate are 330 +/- 25 and 430 +/- 48 microM for the cytosolic and plastid isozymes, respectively. The corresponding Km values for 2-phosphoglycerate are 60 +/- 10 and 112 +/- 22 microM. The two isozymes also have different pH optima and heat labilities. Neither isozyme requires 2,3-bisphosphoglycerate or a divalent cation and neither is regulated by metabolites.     

Construction of a 3-chlorobiphenyl-utilizing recombinant from an intergeneric mating.

Recombinant Pseudomonas sp. strain CB15, which grows on 3-chlorobiphenyl (3CB), was constructed from Pseudomonas sp. strain HF1, which grows on 3-chlorobenzoate, and from Acinetobacter sp. strain P6, which grows on biphenyl, by using a continuous amalgamated culture apparatus. DNA from strains CB15 and HF1 hybridized very strongly to each other, while hybridization between both parental strains, HF1 and P6, was negligible. However, DNA from the recombinant CB15 hybridized moderately to strongly with three specific fragments of parental strain P6. Strains HF1 and P6 did not grow on 3CB, but recombinant strain CB15 mineralized this compound and released inorganic chloride. When growing on 3CB, strain CB15 accumulated brown products, one of which was identified as 3-chloro-5-(2'-hydroxy-3'-chlorophenyl)-1,2-benzoquinone by mass spectrometry. Emulsification and mechanical fragmentation greatly increased the rate of 3CB mineralization by strain CB15. At least three methods of inhibition from catecholic intermediates may account for slow growth on 3CB. The meta fission of 2,3-dihydroxybiphenyl (the nonchlorinated analog of the metabolic intermediate 3-chloro-2',3'-dihydroxybiphenyl) was affected by substrate inhibition (Vmax = 359 nmol.min-1.mg-1, Km = 114 microM, Kss [the inhibition constant] = 951 microM) and was also inhibited by 3-chlorocatechol. The ortho fission of 3-chlorocatechol, a degradation product, followed Michaelis-Menten kinetics (Vmax = 365 nmol.min-1.mg-1, Km = 1 microM), but the addition of 2,3-dihydroxybiphenyl inhibited the reaction (Ki = 0.87 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of meta-cleavage compound hydrolase from a carbazole degrader Pseudomonas resinovorans strain CA10

2-Hydroxy-6-oxo-6-(2'-aminophenyl)-hexa-2,4dienoic acid [6-(2'-aminophenyl)-HODA] hydrolase, involved in carbazole degradation by Pseudomonas resinovorans strain CA10, was purified to near homogeneity from an overexpressing Escherichia coli strain. The enzyme was dimeric, and its optimum pH was 7.0-7.5. Phylogenetic analysis showed the close relationship of this enzyme to other hydrolases involved in the degradation of monocyclic aromatic compounds, and this enzyme was specific for 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (6-phenyl-HODA), having little activity toward 2-hydroxy-6-oxohepta-2,4-dienoic acid and 2-hydroxymuconic semialdehyde. The enzyme had a Km of 2.51 microM and k(cat) of 2.14 (s(-1)) for 6-phenyl-HODA (50 mM sodium phosphate, pH 7.5, 25 degrees C). The effect of the presence of an amino group or hydroxyl group at the 2'-position of phenyl moiety of 6-phenyl-HODA on the enzyme activity was found to be small; the activity decreased only in the order of 6-(2'-aminophenyl)-HODA (2.44 U/mg) > 6-phenyl-HODA (1.99 U / mg) > 2-hydroxy-6-oxo-6-(2'-hydroxyphenyl)-hexa-2,4-dienoic acid (1.05 U/mg). The effects of 2'-substitution on the activity were in accordance with the predicted reactivity based on the calculated lowest unoccupied molecular orbital energy for these substrates.     

Characterization of three distinct extradiol dioxygenases involved in mineralization of dibenzofuran by Terrabacter sp. strain DPO360.

The dibenzofuran-degrading bacterial strain DPO360 represents a new species of the genus Terrabacter together with the previously described dibenzofuran-mineralizing bacterial strain DPO1361 (K.-H. Engesser, V. Strubel, K. Christoglou, P. Fischer, and H. G. Rast, FEMS Microbiol. Lett. 65:205-210, 1989; V. Strubel, Ph.D. thesis, University of Stuttgart, Stuttgart, Germany, 1991; V. Strubel, H. G. Rast, W. Fietz, H.-J. Knackmuss, and K.-H. Engesser, FEMS Microbiol. Lett. 58:233-238, 1989). Two 2,3-dihydroxybiphenyl-1,2-dioxygenases (BphC1 and BphC2) and one catechol-2,3-dioxygenase (C23O) were shown to be expressed in Terrabacter sp. strain DPO360 growing with dibenzofuran as a sole source of carbon and energy. These enzymes exhibited strong sensitivity to oxygen. They were purified to apparent homogeneity as homodimers (BphC and BphC2) and as a homotetrameric catechol-2,3-dioxygenase (C23O). According to their specificity constants kcat/Km, both BphC1 and BphC2 were shown to be responsible for the cleavage of 2,2',3-trihydroxybiphenyl, the first metabolite in dibenzofuran mineralization along the angular dioxygenation pathway. With this substrate, BphC2 exhibited a considerably higher kcat/Km, value (183 microM/min) than BphC1 (29 microM/min). Catechol-2,3-dioxygenase was recognized to be not involved in the ring cleavage of 2,2',3-trihydroxybiphenyl (kcat/Km, 1 microM/min). Analysis of deduced amino acid sequence data of bphC1 revealed 36% sequence identity to nahC from Pseudomonas putida PpG7 (S. Harayama and M. Rekik, J. Biol. Chem. 264:15328-15333, 1989) and about 40% sequence identity to various bphC genes from different Pseudomonas and Rhodococcus strains. In addition, another 2,3-dihydroxybiphenyl-1,2-dioxygenase gene (bphC3) was cloned from the genome of Terrabacter sp. strain DPO360. Expression of this gene, however, could not be detected in Terrabacter sp. strain DPO360 after growth with dibenzofuran.     

The phosphoenolpyruvate carboxykinase of Trypanosoma (Schizotrypanum) cruzi epimastigotes: molecular, kinetic, and regulatory properties

The phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49) of the epimastigote form of Trypanosoma (Schizotrypanum) cruzi has been purified to homogeneity. The enzyme is composed of two apparently identical 42,000 +/- 500 subunits, is highly specific for adenine nucleotides, and has a strict requirement of Mn2+ ions for activity; the activation of the enzyme by ionic Mn2+ reveals that one Mn2+ ion required for each 42,000 subunit. Hyperbolic kinetics are observed for all substrates in the carboxylation reaction with Km (phosphoenolpyruvate) of 0.36 +/- 0.08 mM, Km (HCO-3) of 3.7 +/- 0.2 mM, and Km (Mg-ADP) of 39 +/- 1 microM. In the decarboxylation reaction the kinetics with respect to oxalacetic acid are also hyperbolic with a Km of 27 +/- 3 microM, but towards Mg-ATP there is a biphasic response: hyperbolic at low (less than 250 microM) concentrations with a Km of 39 +/- 1 microM, but at higher concentrations the nucleotide produces a strong inhibition of the enzyme activity. This inhibition is also observed with Mg-GTP and Mg-ITP which are not substrates of the reaction. The results are consistent with an important regulatory function of the enzyme in the amino-acid catabolism of T. cruzi.     

Isolation and characterization of a rhodococcus species strain able to grow on ortho- and para-xylene

Rhodococcus sp. strain YU6 was isolated from soil for the ability to grow on o-xylene as the sole carbon and energy source. Unlike most other o-xylene-degrading bacteria, YU6 is able to grow on p-xylene. Numerous growth substrate range experiments, in addition to the ring-cleavage enzyme assay data, suggest that YU6 initially metabolizes o- and p-xylene by direct aromatic ring oxidation. This leads to the formation of dimethylcatechols, which was further degraded largely through meta-cleavage pathway. The gene encoding meta-cleavage dioxygenase enzyme was PCR cloned from genomic YU6 DNA using previously known gene sequence data from the o-xylene-degrading Rhodococcus sp. strain DK17. Subsequent sequencing of the 918-bp PCR product revealed a 98% identity to the gene, encoding methylcatechol 2,3-dioxygenase from DK17. PFGE analysis followed by Southern hybridization with the catechol 2,3-dioxygenase gene demonstrated that the gene is located on an approximately 560-kb megaplasmid, designated pJYJ1.     

Ketopantoic acid reductase of Pseudomonas maltophilia 845. Purification, characterization, and role in pantothenate biosynthesis

Ketopantoic acid reductase (EC 1.1.1.169), an enzyme that catalyzes the formation of D-(-)-pantoic acid from ketopantoic acid, was purified 6,000-fold to apparent homogeneity with a 35% overall recovery from Pseudomonas maltophilia 845 and then crystallized. The relative molecular mass of the native enzyme, as estimated by the sedimentation equilibrium method, is 87,000 +/- 5,000, and the subunit molecular mass is 30,500. The enzyme shows high specificity for ketopantoic acid as a substrate (Km = 400 microM, Vm = 1,310 units/mg of protein) and NADPH as a coenzyme (Km = 31.8 microM). Only 2-keto-3-hydroxyisovalerate (Km = 8.55 mM, Vm = 35.8 units/mg) was reduced among a variety of other carbonyl compounds tested. The reaction is reversible (Km for D-(-)-pantoic acid = 52.1 mM), although the reaction equilibrium greatly favors the direction of D-(-)-pantoic acid formation. That the enzyme is responsible for the synthesis of D-(-)-pantoic acid necessary for the biosynthesis of pantothenic acid in P. maltophilia 845 is indicated by the observations that only this enzyme is missing in D-(-)-pantoate (or pantothenate)-requiring mutants derived from P. maltophilia 845 among several enzymes (i.e. ketopantoyl lactone reductase (EC 1.1.1.168) and acetohydroxy acid isomeroreductase (EC 1.1.1.86], which may be concerned in the formation of D-(-)-pantoic acid, assayed, whereas it is present in substantial amounts in the parent strain and in spontaneous revertants of the mutants.