Cloning of a Sphingomonas paucimobilis SYK-6 gene encoding a novel oxygenase that cleaves lignin-related biphenyl and characterization of the enzyme

Sphingomonas paucimobilis SYK-6 transforms 2,2'-dihydroxy-3,3'-dimethoxy-5,5'-dicarboxybiphenyl (DDVA), a lignin-related biphenyl compound, to 5-carboxyvanillic acid via 2,2',3-trihydroxy-3'-methoxy-5,5'-dicarboxybiphenyl (OH-DDVA) as an intermediate (15). The ring fission of OH-DDVA is an essential step in the DDVA degradative pathway. A 15-kb EcoRI fragment isolated from the cosmid library complemented the growth deficiency of a mutant on OH-DDVA. Subcloning and deletion analysis showed that a 1.4-kb DNA fragment included the gene responsible for the ring fission of OH-DDVA. An open reading frame encoding 334 amino acids was identified and designated ligZ. The deduced amino acid sequence of LigZ had 18 to 21% identity with the class III extradiol dioxygenase family, including the beta subunit (LigB) of protocatechuate 4,5-dioxygenase of SYK-6 (Y. Noda, S. Nishikawa, K.-I. Shiozuka, H. Kadokura, H. Nakajima, K. Yano, Y. Katayama, N. Morohoshi, T. Haraguchi, and M. Yamasaki, J. Bacteriol. 172:2704-2709, 1990), catechol 2,3-dioxygenase I (MpcI) of Alcaligenes eutrophus JMP222 (M. Kabisch and P. Fortnagel, Nucleic Acids Res. 18:3405-3406, 1990), the catalytic subunit of the meta-cleavage enzyme (CarBb) for 2'-aminobiphenyl-2,3-diol from Pseudomonas sp. strain CA10 (S. I. Sato, N. Ouchiyama, T. Kimura, H. Nojiri, H. Yamane, and T. Omori, J. Bacteriol. 179:4841-4849, 1997), and 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB) of Escherichia coli (E. L. Spence, M. Kawamukai, J. Sanvoisin, H. Braven, and T. D. H. Bugg, J. Bacteriol. 178:5249-5256, 1996). The ring fission product formed from OH-DDVA by LigZ developed a yellow color with an absorption maximum at 455 nm, suggesting meta cleavage. Thus, LigZ was concluded to be a ring cleavage extradiol dioxygenase. LigZ activity was detected only for OH-DDVA and 2,2',3,3'-tetrahydroxy-5,5'-dicarboxybiphenyl and was dependent on the ferrous ion.     

Purification of 2,3-dihydroxybiphenyl 1,2-dioxygenase from Pseudomonas putida OU83 and characterization of the gene (bphC)

The 2,3-dihydroxybiphenyl 1,2-dioxygenase (2,3-DBPD) of Pseudomonas putida OU83 was constitutively expressed and purified to apparent homogeneity. The apparent molecular mass of the native enzyme was 256 kDa, and the subunit molecular mass was 32 kDa. The data suggested that 2,3-DBPD was an octamer of identical subunits. The nucleotide sequence of a DNA fragment containing the bphC region was determined. The deduced protein sequence for 2,3-DBPD consisted of 292 amino acid residues, with a calculated molecular mass of 31.9 kDa, which was in agreement with data for the purified 2,3-DBPD. Nucleotide and amino acid sequence analyses of the bphC gene and its product, respectively, revealed that there was a high degree of homology between the OU83 bphC gene and the bphC genes of Pseudomonas cepacia LB400 and Pseudomonas pseudoalcaligenes KF707.     

Analysis of 66 kDa toxin from Bacillus thuringiensis subsp. kurstaki reveals differential amino terminal processing of protoxin by endogenous protease(s)

The endogenous protease(s) activated crystal toxin from Bacillus thuringiensis subsp. kurstaki was purified and examined. The purified toxin was homogenous, as demonstrated by two-dimensional polyacrylamide gel electrophoresis and contained 1.38 mumoles neutral sugar and 9 nmoles sialic acid per mg protein amino terminal amino acid sequence data revealed that the toxin is a cleavage product of 132 kDa protoxin with glutamic acid-30 of the deduced amino acid sequence of the crystal protein (Schnepf, H.E., Wong, H.C. and Whiteley, H.R. (1985) J. Biol. Chem. 260: 6264-6272) at the amino terminus.     

2-Ketocyclohexanecarboxyl coenzyme A hydrolase, the ring cleavage enzyme required for anaerobic benzoate degradation by Rhodopseudomonas palustris

2-Ketocyclohexanecarboxyl coenzyme A (2-ketochc-CoA) hydrolase has been proposed to catalyze an unusual hydrolytic ring cleavage reaction as the last unique step in the pathway of anaerobic benzoate degradation by bacteria. This enzyme was purified from the phototrophic bacterium Rhodopseudomonas palustris by sequential Q-Sepharose, phenyl-Sepharose, gel filtration, and hydroxyapatite chromatography. The sequence of the 25 N-terminal amino acids of the purified hydrolase was identical to the deduced amino acid sequence of the badI gene, which is located in a cluster of genes involved in anaerobic degradation of aromatic acids. The deduced amino acid sequence of badI indicates that 2-ketochc-CoA hydrolase is a member of the crotonase superfamily of proteins. Purified BadI had a molecular mass of 35 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a native molecular mass of 134 kDa as determined by gel filtration. This indicates that the native form of the enzyme is a homotetramer. The purified enzyme was insensitive to oxygen and catalyzed the hydration of 2-ketochc-CoA to yield pimelyl-CoA with a specific activity of 9.7 micromol min(-1) mg of protein(-1). Immunoblot analysis using polyclonal antiserum raised against the purified hydrolase showed that the synthesis of BadI is induced by growth on benzoate and other proposed benzoate pathway intermediates but not by growth on pimelate or succinate. An R. palustris mutant, carrying a chromosomal disruption of badI, did not grow with benzoate and other proposed benzoate pathway intermediates but had wild-type doubling times on pimelate and succinate. These data demonstrate that BadI, the 2-ketochc-CoA hydrolase, is essential for anaerobic benzoate metabolism by R. palustris.     

Purification of the cyclooxygenase that forms prostaglandins. Demonstration of two forms of iron in the holoenzyme

The fatty acid cyclooxygenase (ec 1.14.99.1) that produces the prostaglandin and thromboxane precursor, 15-hydroperoxy-9 alpha, 11 alpha-peroxidoprosta-5, 13-dienoic acid (PGG2), has been purified from sheep vesicular glands to a specific activity of 46,000 units/mg of protein by combining detergent solubilization, (NH4)SO4 fractionation, chromatography on DEAE-cellulose and Flurbiprofen-Sepharose, isoelectric focusing, and gel filtration. The final enzyme preparation exhibited only one band of 70,000 molecular weight following sodium dodecyl sulfate gel electrophoresis and staining with Coomassie blue. Treatment of the purified oxygenase with [3H] acetylsalicylic acid yielded a radioactive product which co-electrophoresed with the protein of 70,000 molecular weight. Thus, the isolated protein appeared to be the same one which, in crude preparations, selectively binds acetyl groups in association with prostaglandin synthetic activity. Incubation of the purified oxygenase with [1-14C] arachidonic acid in the presence of stannous chloride yielded only 9 alpha, 11 alpha, 15-trihydroxy-prosta-5,13-dienoic acid (PGF2alpha). Without stannous chloride, a mixture of radioactive products was observed which was characteristic of nonenzymic breakdown of PGG2. Thus, the isolated enzyme catalyzed the insertion of both oxygen molecules required for the formation of prostaglandins and thromboxanes from polyunsaturated fatty acid substrates. The aerobic absorption spectrum of the isolated oxygenase showed a faint peak at 412 nm indicative of heme. The iron content indicated that a significant amount of nonheme iron was present. The purified oxygenase was activated by added hemin, which was readily bound to the protein. The subsequently isolated heme-protein complex showed a major absorption peak at 407 nm.     

Expression, purification, and properties of recombinant barley (Hordeum sp.) hemoglobin. Optical spectra and reactions with gaseous ligands

A cDNA encoding barley hemoglobin (Hb) has been cloned into pUC 19 and expressed in Escherichia coli. The resulting fusion protein has five extra amino acids at the N terminus compared with the native protein, resulting in a protein of 168 amino acids (18.5 kDa). The recombinant Hb is expressed constitutively. Extracts made from the bacteria containing the recombinant fusion construct contain a protein with a subunit molecular mass of approximately 18.5 kDa comprising approximately 5% total soluble protein. Recombinant Hb was purified to homogeneity according to SDS-polyacrylamide gel electrophoresis by sequential polyethylene glycol precipitation and fast protein liquid chromatography. Its native molecular mass as assessed by fast protein liquid chromatography-size exclusion was 40 kDa suggesting that it is a dimer. Ligand binding experiments demonstrate that 1) barley Hb has a very slow oxygen dissociation rate constant (0.0272 s-1) relative to other Hbs, and 2) the heme of ferrous and ferric forms of the barley Hb is low spin six-coordinate. The subunit structure, optical spectrum, and oxygen dissociation rate of native barley hemoglobin are indistinguishable from those obtained for the recombinant protein. The implications of these kinetic data on the in vivo function of barley Hb are discussed.     

Purification, characterization, and sequence analysis of 2-aminomuconic 6-semialdehyde dehydrogenase from Pseudomonas pseudoalcaligenes JS45

2-Aminonumconic 6-semialdehyde is an unstable intermediate in the biodegradation of nitrobenzene and 2-aminophenol by Pseudomonas pseudoalcaligenes JS45. Previous work has shown that enzymes in cell extracts convert 2-aminophenol to 2-aminomuconate in the presence of NAD+. In the present work, 2-aminomuconic semialdehyde dehydrogenase was purified and characterized. The purified enzyme migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular mass of 57 kDa. The molecular mass of the native enzyme was estimated to be 160 kDa by gel filtration chromatography. The optimal pH for the enzyme activity was 7.3. The enzyme is able to oxidize several aldehyde analogs, including 2-hydroxymuconic semialdehyde, hexaldehyde, and benzaldehyde. The gene encoding 2-aminomuconic semialdehyde dehydrogenase was identified by matching the deduced N-terminal amino acid sequence of the gene with the first 21 amino acids of the purified protein. Multiple sequence alignment of various semialdehyde dehydrogenase protein sequences indicates that 2-aminomuconic 6-semialdehyde dehydrogenase has a high degree of identity with 2-hydroxymuconic 6-semialdehyde dehydrogenases.     

Cloning and sequence analysis of cDNA encoding a putative juvenile hormone esterase from the Colorado potato beetle

In the Colorado potato beetle, Leptinotarsa decemlineata, reproduction and diapause are mediated by the juvenile hormone (JH) titer in the hemolymph. This titer is controlled by JH synthesis in the corpora allata and by JH degradation. The main pathway of JH degradation is by JH esterase in the hemolymph. The native JH esterase appeared to be a dimer consisting of two 57 kDa subunits (Vermunt et al., 1997). The 57 kDa subunit of JH esterase was digested with endoproteinase Lys-C and the digestion products were separated by reversed phase HPLC. Three different peptides were collected and sequenced. The amino acid sequence of one peptide showed high similarity to fragments of other insect esterases. Based on the amino acid sequence of these peptides, degenerate primers were constructed for RT-PCR. A PCR product of 1.3 kb was obtained and sequenced. This product was used to screen a cDNA library for a complete cDNA copy and to analyze the messenger RNA from larvae and adult beetles. The size of the messenger RNA was 1.7 kb. The complete amino acid sequence of the protein was deduced from the nucleotide sequence of overlapping clones from a cDNA library and a 5'RACE product. An open reading frame (ORF) of 1545 base pairs encoded a 57 kDa protein with a predicted pI of 5.5. The ORF contained the sequence of the three peptides. It showed no significant homology to other proteins present in databases, but it did contain several functional esterase motifs.     

2-oxo-1,2-dihydroquinoline 8-monooxygenase: phylogenetic relationship to other multicomponent nonheme iron oxygenases

2-Oxo-1,2-dihydroquinoline 8-monooxygenase, an enzyme involved in quinoline degradation by Pseudomonas putida 86, had been identified as a class IB two-component nonheme iron oxygenase based on its biochemical and biophysical properties (B. Rosche, B. Tshisuaka, S. Fetzner, and F. Lingens, J. Biol. Chem. 270:17836-17842, 1995). The genes oxoR and oxoO, encoding the reductase and the oxygenase components of the enzyme, were sequenced and analyzed. oxoR was localized approximately 15 kb downstream of oxoO. Expression of both genes was detected in a recombinant Pseudomonas strain. In the deduced amino acid sequence of the NADH:(acceptor) reductase component (OxoR, 342 amino acids), putative binding sites for a chloroplast-type [2Fe-2S] center, for flavin adenine dinucleotide, and for NAD were identified. The arrangement of these cofactor binding sites is conserved in all known class IB reductases. A dendrogram of reductases confirmed the similarity of OxoR to other class IB reductases. The oxygenase component (OxoO, 446 amino acids) harbors the conserved amino acid motifs proposed to bind the Rieske-type [2Fe-2S] cluster and the mononuclear iron. In contrast to known class IB oxygenase components, which are composed of differing subunits, OxoO is a homomultimer, which is typical for class IA oxygenases. Sequence comparison of oxygenases indeed revealed that OxoO is more related to class IA than to class IB oxygenases. Thus, 2-oxo-1,2-dihydroquinoline 8-monooxygenase consists of a class IB-like reductase and a class IA-like oxygenase. These results support the hypothesis that multicomponent enzymes may be composed of modular elements having different phylogenetic origins.     

Purification and characterization of nitrobenzene nitroreductase from Pseudomonas pseudoalcaligenes JS45

Pseudomonas pseudoalcaligenes JS45 grows on nitrobenzene as a sole source of carbon, nitrogen, and energy. The catabolic pathway involves reduction to hydroxylaminobenzene followed by rearrangement to o-amino-phenol and ring fission (S. F. Nishino and J. C. Spain, Appl. Environ. Microbiol. 59:2520, 1993). A nitrobenzene-inducible, oxygen-insensitive nitroreductase was purified from extracts of JS45 by ammonium sulfate precipitation followed by anion-exchange and gel filtration chromatography. A single 33-kDa polypeptide was detected by denaturing gel electrophoresis. The size of the native protein was estimated to be 30 kDa by gel filtration. The enzyme is a flavoprotein with a tightly bound flavin mononucleotide cofactor in a ratio of 2 mol of flavin per mol of protein. The Km for nitrobenzene is 5 microM at an initial NADPH concentration of 0.5 mM. The Km for NADPH at an initial nitrobenzene concentration of 0.1 mM is 183 microM. Nitrosobenzene was not detected as an intermediate of nitrobenzene reduction, but nitrosobenzene is a substrate for the enzyme, and the specific activity for nitrosobenzene is higher than that for nitrobenzene. These results suggest that nitrosobenzene is formed but is immediately reduced to hydroxylaminobenzene. Hydroxylaminobenzene was the only product detected after incubation of the purified enzyme with nitrobenzene and NADPH. Hydroxylaminobenzene does not serve as a substrate for further reduction by this enzyme. The products and intermediates are consistent with two two-electron reductions of the parent compound. Furthermore, the low Km and the inducible control of enzyme synthesis suggest that nitrobenzene is the physiological substrate for this enzyme.     

Biochemical and genetic characterization of a gentisate 1, 2-dioxygenase from Sphingomonas sp. strain RW5

A 4,103-bp long DNA fragment containing the structural gene of a gentisate 1,2-dioxygenase (EC 1.13.11.4), gtdA, from Sphingomonas sp. strain RW5 was cloned and sequenced. The gtdA gene encodes a 350-amino-acid polypeptide with a predicted size of 38.85 kDa. Comparison of the gtdA gene product with protein sequences in databases, including those of intradiol or extradiol ring-cleaving dioxygenases, revealed no significant homology except for a low similarity (27%) to the 1-hydroxy-2-naphthoate dioxygenase (phdI) of the phenanthrene degradation in Nocardioides sp. strain KP7 (T. Iwabuchi and S. Harayama, J. Bacteriol. 179:6488-6494, 1997). This gentisate 1,2-dioxygenase is thus a member of a new class of ring-cleaving dioxygenases. The gene was subcloned and hyperexpressed in E. coli. The resulting product was purified to homogeneity and partially characterized. Under denaturing conditions, the polypeptide exhibited an approximate size of 38.5 kDa and migrated on gel filtration as a species with a molecular mass of 177 kDa. The enzyme thus appears to be a homotetrameric protein. The purified enzyme stoichiometrically converted gentisate to maleylpyruvate, which was identified by gas chromatography-mass spectrometry analysis as its methyl ester. Values of affinity constants (Km) and specificity constants (Kcat/Km) of the enzyme were determined to be 15 microM and 511 s-1 M-1 x 10(4) for gentisate and 754 microM and 20 s-1 M-1 x 10(4) for 3, 6-dichlorogentisate. Three further open reading frames (ORFs) were found downstream of gtdA. The deduced amino acid sequence of ORF 2 showed homology to several isomerases and carboxylases, and those of ORFs 3 and 4 exhibited significant homology to enzymes of the glutathione isomerase superfamily and glutathione reductase superfamily, respectively.     

Catabolism of isonicotinate by Mycobacterium sp. INA1: extended description of the pathway and purification of the molybdoenzyme isonicotinate dehydrogenase

Catabolism of isonicotinate by Mycobacterium sp. INA1 has been shown to proceed via 2-hydroxyisonicotinate, 2,6-dihydroxyisonicotinate (citrazinate), citrazyl-CoA and 2,6-dioxopiperidine-4-carboxyl-CoA. An extended pathway involving propane-1,2,3-tricarboxylate as a further intermediate is presented in this paper. Propane-1,2,3-tricarboxylate was oxidized stepwise to 2-oxoglutarate involving an oxidase, aconitase and isocitrate dehydrogenase. Isonicotinate dehydrogenase catalyses the first step of isonicotinate metabolism in Mycobacterium sp. INA1. The enzyme was purified to apparent homogeneity by a three-step procedure. Enrichment was accompanied by partial loss in specific activity. The native enzyme had a molecular mass of either 125 kDa or 250 kDa, when estimated by native gradient PAGE or gel filtration, respectively. SDS-gel electrophoresis revealed three types of subunits with molecular masses of approximately 83, 31 and 19 kDa. N-Terminal amino acid sequences of all three subunits have been determined. Molybdenum, iron, acid-labile sulphur and FAD were present at molar ratios of 1, 4, 4, 1 per protomer (125 kDa). The molybdenum-complexing cofactor was shown to be molybdopterin cytosine dinucleotide. Besides isonicotinate, only quinoline-4-carboxylate was found to be oxidized at appreciable rates.     

Properties and structure of spermidine acetyltransferase in Escherichia coli

Spermidine acetyltransferase (SAT) from Escherichia coli was purified about 40,000-fold. The molecular mass of native SAT was 95 kDa, and it consisted of four identical subunits. The products formed from the reaction of acetyl-CoA with spermidine by SAT were N1- and N8-acetylspermidine. The Km values for acetyl-CoA, spermidine, and spermine were 2 microM, 1.29 mM, and 220 microM, respectively. The enzymatic activity increased by 2.5-3.5-fold under the condition of poor nutrition but not in response to cold shock or high pH. By using synthetic oliogonucleotides deduced from amino acid sequences of the peptides in SAT, a polymerase chain reaction product with a length of 250 nucleotides was obtained. Using this polymerase chain reaction product, the gene encoding SAT (speG) was cloned and mapped at 35.6 min in the E. coli chromosome. E. coli cells transformed with the cloned speG gene increased SAT activity by 8-40-fold. The gene encoded a 186-amino acid protein, but SAT consisted of 185 amino acids because the initiator methionine was liberated from the protein. Thus, the predicted molecular mass was 21,756 Da. Significant similarity to aminoglycoside acetyltransferase and peptide N-acetyltransferase was observed in the amino acid sequence 87-141, and some similarity with spermidine-preferential binding protein (potD protein) in the spermidine-preferential uptake system was observed in the amino acid sequence 122-141. The results suggest that the active center of SAT may be located in the COOH-terminal portion.     

A novel glycerol kinase from Flavobacterium meningosepticum: characterization, gene cloning and primary structure

A thermostable glycerol kinase (FGK) was purified 34-fold to homogeneity from Flavobacterium meningosepticum. The molecular masses of the enzyme were 200 kDa by gel filtration and 50 kDa by SDS-PAGE. The Km for glycerol and ATP were 0.088 and 0.030 mM, respectively. The enzyme was stable at 65 degrees C for 10 min and at 37 degrees C for two weeks. The enzyme gene was cloned into Escherichia coli and its complete DNA was sequenced. The FGK gene consists of an open reading frame of 1494-bp encoding a protein of 498 amino acids. The deduced amino acid sequence of the gene had 40-60% similarity to those of glycerol kinases from other origins and the amino acid sequence of the putative active site residue reported for E. coli GK is identical to the corresponding sequence of FGK except for one amino acid residue.     

Function of the proximal stomach after Nissen fundoplication

A novel chitinase gene was isolated from Metarhizium anisopliae grown in a medium containing chitin as the sole carbon source. Comparisons of nucleotide sequence of the isolated gene with those of other fungal chitinase genes showed low sequence identity (24.4-36.4%) except for the active site of chitinase. In addition, molecular mass determination of the fused gene product separated on a gel showed that the fused chitinase seems to be about 70 kDa, while the molecular mass calculated from the deduced amino acid sequence can be about 58 kDa. These molecular masses were different from values of 33 kDa for an endochitinase and 110 kDa for an exochitinase (N-acetylglucosaminidase) from M. anisopliae published previously.     

The human cytomegalovirus UL74 gene encodes the third component of the glycoprotein H-glycoprotein L-containing envelope complex

The human cytomegalovirus (HCMV) gCIII envelope complex is composed of glycoprotein H (gH; gpUL75), glycoprotein L (gL; gpUL115), and a third, 125-kDa protein not related to gH or gL (M. T. Huber and T. Compton, J. Virol. 71:5391-5398, 1997; L. Li, J. A. Nelson, and W. J. Britt, J. Virol. 71:3090-3097, 1997). Glycosidase digestion analysis demonstrated that the 125-kDa protein was a glycoprotein containing ca. 60 kDa of N-linked oligosaccharides on a peptide backbone of 65 kDa or less. Based on these biochemical characteristics, two HCMV open reading frames, UL74 and TRL/IRL12, were identified as candidate genes for the 125-kDa glycoprotein. To identify the gene encoding the 125-kDa glycoprotein, we purified the gCIII complex, separated the components by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and subjected gH and the 125-kDa glycoprotein to amino acid microsequence analysis. Microsequencing of an internal peptide derived from purified 125-kDa glycoprotein yielded the amino acid sequence LYVGPTK. A FASTA search revealed an exact match of this sequence to amino acids 188 to 195 of the predicted product of the candidate gene UL74, which we have designated glycoprotein O (gO). Anti-gO antibodies reacted in immunoblots with a protein species migrating at ca. 100 to 125 kDa in lysates of HCMV-infected cells and with 100- and 125-kDa protein species in purified virions. Anti-gO antibodies also immunoprecipitated the gCIII complex and recognized the 125-kDa glycoprotein component of the gCIII complex. Positional homologs of the UL74 gene were found in other betaherpesviruses, and comparisons of the predicted products of the UL74 homolog genes demonstrated a number of conserved biochemical features.