The protein encoded by the Shewanella colwellianamelA gene is a p-hydroxyphenylpyruvate dioxygenase

Abstract The identity of the product of the melA gene from Shewanella colelliana with the enzyme p -hydroxyphenylpyruvic dioxygenase is shown. Cloning of the melA gene endowed Escherichia coli with the capacity to synthesize melanin-like pigments from L-tyrosine. E. coli contained transaminases that transforms L-tyrosine into p -hydroxyphenylpyruvate. This keto acid was detected in the cultures. On the other hand, E. coli containing melA was able to go further in the catabolic pathway, releasing a great amount of homogentisic acid. This acid can spontaneously polymerize giving the pigment. Furthermore, p -hydroxyphenylpyruvate dioxygenase activity was detected in this system. Analysis of the deduced amino acid sequence revealed a high homology with the p -hydroxyphenylpyruvate deoxygenase enzyme from different organisms.     

Phosphoglycerate mutase from Streptomyces coelicolor A3(2): purification and characterization of the enzyme and cloning and sequence analysis of the gene.

The enzyme 3-phosphoglycerate mutase was purified 192-fold from Streptomyces coelicolor, and its N-terminal sequence was determined. The enzyme is tetrameric with a subunit Mr of 29,000. It is 2,3-bisphosphoglycerate dependent and inhibited by vanadate. The gene encoding the enzyme was cloned by using a synthetic oligonucleotide probe designed from the N-terminal peptide sequence, and the complete coding sequence was determined. The deduced amino acid sequence is 64% identical to that of the phosphoglycerate mutase of Saccharomyces cerevisiae and has substantial identity to those of other phosphoglycerate mutases.     

Cloning and sequencing of an intracellular D(-)-3-hydroxybutyrate oligomer hydrolase from Acidovorax sp. strain SA1 and purification of the enzyme

The gene of an intracellular D(-)-3-hydroxybutyrate oligomer hydrolase (i3HBOH) was cloned and sequenced from a poly(3-hydroxybutyrate) (PHB)-degrading bacterium, Acidovorax sp. strain SA1. The i3HBOH gene has 876 nucleotides corresponding to the deduced sequence of 292 amino acids. In this amino acid sequence, the general lipase box sequence (G-X₁-S-X₂-G) was found, whose serine residue was determined to the active sites serine by site-directed mutagenesis. An i3HBOH was purified to electrophoretical homogeneity from SA1. The molecular mass of the purified enzyme was estimated to be 32 kDa by SDS-PAGE. The N-terminal amino acid sequence of the purified enzyme corresponded to the deduced N-terminal amino acid sequence in the cloned i3HBOH gene. This is the first cloning and sequencing of an intracellular D(-)-3-hydroxybutyrate oligomer hydrolase gene to date. Received: 19 October 2001 / Accepted: 7 December 2001     

The nucleotide sequence of the tyrosinase gene from Streptomyces antibioticus and characterization of the gene product

The sequence of a 1.56-kb DNA fragment containing the tyrosinase gene (mel) from Streptomyces antibioticus was determined and the Mr (30612) and amino acid (aa) sequence of the protein were deduced from the nucleotide (nt) sequence. Intracellular and extracellular tyrosinase from S. antibioticus, transformed with pIJ702 (containing mel), were purified to homogeneity; the Mr (29 500), as determined by Sephadex G-75 chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was consistent with the value derived from the nt sequence. Edman degradation established that the N-terminal sequence of both the intracellular and extracellular forms of tyrosinase are identical and correspond to the aa sequence derived from the structural gene. In addition, this sequence exhibits striking homology to the N-terminal region of the intracellular and extracellular enzyme purified from Streptomyces glaucescens (Crameri et al., 1982). An additional open reading frame (ORF438) upstream of the mel gene, was also identified that appears to code for a protein (Mr = 14 754) with a putative signal sequence.     

Genetic and biochemical characterization of the Lactobacillus delbrueckii subsp. lactis bacteriophage LL-H lysin.

LL-H, a virulent phage of Lactobacillus delbrueckii subsp. lactis, produces a peptidoglycan-degrading enzyme, Mur, that is effective on L. delbrueckii, Lactobacillus acidophilus, Lactobacillus helveticus, and Pediococcus damnosus cell walls. In this study, the LL-H gene mur was cloned into Escherichia coli, its nucleotide sequence was determined, and the enzyme produced in E. coli was purified and biochemically characterized. Mur was purified 112-fold by means of ammonium sulfate precipitation and cation-exchange chromatography. The cell wall-hydrolyzing activity was found to be associated with a 34-kDa protein. The C-terminal domain of Mur is not essential for catalytic activity since it can be removed without destroying the lytic activity. The N-terminal sequence of the purified lysin was identical to that deduced from the nucleotide sequence, but the first methionine is absent from the mature protein. The N-terminal part of this 297-amino-acid protein had homology with several Chalaropsis-type lysozymes. Reduction of purified and Mur-digested L. delbrueckii cell wall material with labeled NaB3H4 indicated that the enzyme is a muramidase. The temperature optimum of purified Mur is between 30 and 40 degrees C, and the pH optimum is around 5.0. The LL-H lysin Mur is stable at temperatures below 60 degrees C.     

Cloning, sequencing, and expression of the meso-diaminopimelate dehydrogenase gene from Bacillus sphaericus

The gene encoding the meso-diaminopimelate dehydrogenase of Bacillus sphaericus was cloned into E. coli cells and its complete DNA sequence was determined. The meso-diaminopimelate dehydrogenase gene consisted of 978 nucleotides and encoded 326 amino acid residues corresponding to the subunit of the dimeric enzyme. The amino acid sequence deduced from the nucleotide sequence of the enzyme gene of B. sphaericus showed 50% identity with those of the enzymes from Corynebacterium glutamicum and Brevibacterium flavum. The enzyme gene from B. sphaericus was highly expressed in E. coli cells. We purified the enzyme to homogeneity from a transformant with 76% recovery. The N-terminal amino acid of both the enzyme from B. sphaericus and the transformant were serine, indicating that the N-terminal methionine is removed by post-translational modification in B. sphaericus and E. coli cells.     

The HtrA (DegP) protein, essential for Escherichia coli survival at high temperatures, is an endopeptidase.

As a preliminary step in the understanding of the function of the Escherichia coli HtrA (DegP) protein, which is indispensable for bacterial survival only at elevated temperatures, the protein was purified and partially characterized. The HtrA protein was purified from cells carrying the htrA gene cloned into a multicopy plasmid, resulting in its overproduction. The sequence of the 13 N-terminal amino acids of the purified HtrA protein was determined and was identical to the one predicted for the mature HtrA protein by the DNA sequence of the cloned gene. Moreover, the N-terminal sequence showed that the 48-kilodalton HtrA protein is derived by cleavage of the first 26 amino acids of the pre-HtrA precursor polypeptide and that the point of cleavage follows a typical target sequence recognized by the leader peptidase enzyme. The HtrA protein was shown to be a specific endopeptidase which was inhibited by diisopropylfluorophosphate, suggesting that HtrA is a serine protease.     

Molecular cloning and nucleotide sequence of the gene encoding a H2O2-forming NADH oxidase from the extreme thermophilic Thermus thermophilus HB8 and its expression in Escherichia coli.

The sequence of the 32 N-terminal amino acids of the NADH oxidase from the extreme thermophile, Thermus thermophilus HB8, was used to synthesize oligonucleotides to probe for the respective gene in a genomic library of T. thermophilus HB8. The gene encoding the NADH oxidase, designated nox, was cloned, its nucleotide sequence was determined and found to be colinear with the N-terminal sequence of the enzyme. The molecular mass of 26835 Da, as deduced from the nox gene, agrees with that of the purified NADH oxidase from T. thermophilus HB8 (25,000 Da), as estimated by polyacrylamide gel electrophoresis under denaturing conditions. The nox gene was overexpressed in Escherichia coli and a protocol for the rapid purification of the enzyme was developed. The E. coli-borne T. thermophilus HB8 NADH oxidase has properties identical to those of the authentic T. thermophilus HB8 enzyme and possesses a high thermal stability.     

Biosynthesis of riboflavin: cloning, sequencing, mapping, and expression of the gene coding for GTP cyclohydrolase II in Escherichia coli.

GTP cyclohydrolase II catalyzes the first committed step in the biosynthesis of riboflavin. The gene coding for this enzyme in Escherichia coli has been cloned by marker rescue. Sequencing indicated an open reading frame of 588 bp coding for a 21.8-kDa peptide of 196 amino acids. The gene was mapped to a position at 28.2 min on the E. coli chromosome and is identical with ribA. GTP cyclohydrolase II was overexpressed in a recombinant strain carrying a plasmid with the cloned gene. The enzyme was purified to homogeneity from the recombinant strain. The N-terminal sequence determined by Edman degradation was identical to the predicted sequence. The sequence is homologous to the 3' part of the central open reading frame in the riboflavin operon of Bacillus subtilis.     

Purification and characterization of the S-adenosylmethionine:glutamyl methyltransferase that modifies membrane chemoreceptor proteins in bacteria

The enzyme (EC 2.1.1.24) from Salmonella typhimurium that catalyzes the S-adenosylmethionine-dependent methyl esterification of glutamyl residues in membrane chemoreceptor proteins has been purified to homogeneity, and the nucleotide sequence of the gene coding for this protein, cheR, has been determined. The molecular weight, amino acid composition, and N-terminal amino acid sequence of the purified protein correspond to the values predicted from the sequence of the gene. The pure protein is a 33-kDa monomer. Kinetic studies indicate that, at levels of receptor and S-adenosylmethionine present in wild type cells, the transferase is nearly saturated. The enzyme has a relatively low turnover number, approximately 10 mol of methylester formed per mol of enzyme per min; and there appear to be only approximately 200 methyltransferase monomers per wild type cell.     

Novel prolyl tri/tetra-peptidyl aminopeptidase from Streptomyces mobaraensis: substrate specificity and enzyme gene cloning

The prolyl peptidase that removes the tetra-peptide of pro-transglutaminase was purified from Streptomyces mobaraensis mycelia. The substrate specificity of the enzyme using synthetic peptide substrates showed proline-specific activity with not only tripeptidyl peptidase activity, but also tetrapeptidyl peptidase activity. However, the enzyme had no other exo- and endo-activities. This substrate specificity is different from proline specific peptidases so far reported. The enzyme gene was cloned, based on the direct N-terminal amino acid sequence of the purified enzyme, and the entire nucleotide sequence of the coding region was determined. The deduced amino acid sequence revealed an N-terminal signal peptide sequence (33 amino acids) followed by the mature protein comprising 444 amino acid residues. This enzyme shows no remarkable homology with enzymes belonging to the prolyl oligopeptidase family, but has about 65% identity with three tripeptidyl peptidases from Streptomyces lividans, Streptomyces coelicolor, and Streptomyces avermitilis. Based on its substrate specificity, a new name, "prolyl tri/tetra-peptidyl aminopeptidase," is proposed for the enzyme.     

The primary structure of Escherichia coli L-threonine dehydrogenase

The complete primary structures of Escherichia coli L-threonine dehydrogenase has been deduced by sequencing the cloned tdh gene. The primary structure so determined agrees with results obtained independently for the amino acid composition, the N-terminal amino acid sequence (20 residues), and a short sequence at the end of an internal peptide of the purified enzyme. The presence of a predicted Asp-Pro bond at residues 148 and 149 was confirmed by treatment of purified threonine dehydrogenase with dilute acid and subsequent analysis of the resulting cleavage products. The primary structure of L-threonine dehydrogenase from E. coli has been examined for possible homology to other NAD+-dependent dehydrogenases; indications are that this enzyme is a member of the zinc-containing long-chain alcohol/polyol dehydrogenase family.     

Nucleotide sequence and genetic structure of a novel carbaryl hydrolase gene (cehA) from Rhizobium sp. strain AC100

Rhizobium sp. strain AC100, which is capable of degrading carbaryl (1-naphthyl-N-methylcarbamate), was isolated from soil treated with carbaryl. This bacterium hydrolyzed carbaryl to 1-naphthol and methylamine. Carbaryl hydrolase from the strain was purified to homogeneity, and its N-terminal sequence, molecular mass (82 kDa), and enzymatic properties were determined. The purified enzyme hydrolyzed 1-naphthyl acetate and 4-nitrophenyl acetate indicating that the enzyme is an esterase. We then cloned the carbaryl hydrolase gene (cehA) from the plasmid DNA of the strain and determined the nucleotide sequence of the 10-kb region containing cehA. No homologous sequences were found by a database homology search using the nucleotide and deduced amino acid sequences of the cehA gene. Six open reading frames including the cehA gene were found in the 10-kb region, and sequencing analysis shows that the cehA gene is flanked by two copies of insertion sequence-like sequence, suggesting that it makes part of a composite transposon.     

Purification of Cytochrome P-450«/A from n-Alkane-grown Cells of Candida maltosa,and Cloning and Nucleotide Sequencing of the Encoding Gene

When the cells of an n-alkane-assimilating yeast, Candida maltosa I AM12247, were transferred from a glucose medium to an n-alkane medium, various enzymes are induced in the endoplasmic reticulum and peroxisome. Cytochrome P-450alk, one of these enzymes in the endoplasmic reticulum, was purified after mild solubilization of the membrane, followed by a few steps of chromatography. The enzyme was characterized spectrophotometrically and its N-terminal amino acid sequence (12 residues) was determined.

Using oligonucleotide probes prepared to match parts of the N-terminal amino acid sequence and of the partial cDNA sequence of cytochrome P-450alk of C. maltosa EH 15, we isolated from a gene library of C. maltosa I AM 12247 a clone which had a gene encoding cytochrome P-450a/Ar. By nucleotide sequencing of this gene, the amino acid sequence of this enzyme was deduced. It consisted of 523 amino acids (59,838 daltons), with a non-cleavable signal sequence in the N-terminal region. The structure of this enzyme was compared with some other members of the cytochrome P-450 superfamily.     

Isolation and characterization of the gene encoding gluconolactonase from Zymomonas mobilis.

The gene encoding the enzyme gluconolactonase (D-glucono-delta-lactone lactonohydrolase, EC 3.1.1.17) has been isolated from a recombinant library of genomic Zymomonas mobilis DNA, by detection of enzyme activity in recombinant clones. The gene encoded a protein of 320 amino acids, which is processed to the mature enzyme of 285 amino acids (31079 Da) by cleavage at an Ala-Ala bond, as determined from N-terminal sequencing of the purified enzyme. A minor sequence commencing at amino acid 6 is suggestive of an alternative start of translation at the ATG codon of amino acid 5; in this case the expressed enzyme would remain cytoplasmic, whereas it is presumed that the main portion is directed to the membrane of periplasm by the leader sequence.     

The desymmetrization of bicyclic beta -diketones by an enzymatic retro-Claisen reaction. A new reaction of the crotonase superfamily

The enzyme 6-oxocamphor hydrolase, which catalyzes the desymmetrization of 6-oxocamphor to yield (2R,4S)-alpha-campholinic acid, has been purified with a factor of 35.7 from a wild type strain of Rhodococcus sp. NCIMB 9784 grown on (1R)-(+)-camphor as the sole carbon source. The enzyme has a subunit molecular mass of 28,488 Da by electrospray mass spectrometry and a native molecular mass of approximately 83,000 Da indicating that the active protein is trimeric. The specific activity was determined to be 357.5 units mg(-)1, and the K(m) was determined to be 0.05 mm for the natural substrate. The N-terminal amino acid sequence was obtained from the purified protein, and using this information, the gene encoding the enzyme was cloned. The translation of the gene was found to bear significant homology to the crotonase superfamily of enzymes. The gene is closely associated with an open reading frame encoding a ferredoxin reductase that may be involved in the initial step in the biodegradation of camphor. A mechanism for 6-oxocamphor hydrolase based on sequence homology and the known mechanism of the crotonase enzymes is proposed.