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Identification, Cloning, and Characterization of l-Phenylserine Dehydrogenase from Pseudomonas syringae NK-15.

Ueshima S, Muramatsu H, Nakajima T, Yamamoto H, Kato S, Misono H, Nagata S - Enzyme Res (2010)

Bottom Line: The purified ORF3 enzyme showed l-phenylserine dehydrogenase activity.The genes encoding l-phenylserine dehydrogenase and d-phenylserine dehydrogenase, which is induced by phenylserine, are located in a single operon.The reaction products of both enzymatic reactions were 2-aminoacetophenone and CO(2).

View Article: PubMed Central - PubMed

Affiliation: The United Graduate School of Agricultural Science, Ehime University, 3-5-7 Tarumi, Matsuyama 790-8566, Japan.

ABSTRACT
The gene encoding d-phenylserine dehydrogenase from Pseudomonas syringae NK-15 was identified, and a 9,246-bp nucleotide sequence containing the gene was sequenced. Six ORFs were confirmed in the sequenced region, four of which were predicted to form an operon. A homology search of each ORF predicted that orf3 encoded l-phenylserine dehydrogenase. Hence, orf3 was cloned and overexpressed in Escherichia coli cells and recombinant ORF3 was purified to homogeneity and characterized. The purified ORF3 enzyme showed l-phenylserine dehydrogenase activity. The enzymological properties and primary structure of l-phenylserine dehydrogenase (ORF3) were quite different from those of d-phenylserine dehydrogenase previously reported. l-Phenylserine dehydrogenase catalyzed the NAD(+)-dependent oxidation of the β-hydroxyl group of l-β-phenylserine. l-Phenylserine and l-threo-(2-thienyl)serine were good substrates for l-phenylserine dehydrogenase. The genes encoding l-phenylserine dehydrogenase and d-phenylserine dehydrogenase, which is induced by phenylserine, are located in a single operon. The reaction products of both enzymatic reactions were 2-aminoacetophenone and CO(2).

No MeSH data available.


Related in: MedlinePlus

Genetic map around the d-phenylserine dehydrogenase gene. orf1: putative transcriptional regulator gene, orf2: putative MFS transporter gene, orf3: l-phenylserine dehydrogenase gene, orf4: putative dihydroxy acid dehydratase gene, orf5: d-phenylserine dehydrogenase gene, orf6: putative ABC peptide transporter gene.
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Related In: Results  -  Collection


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fig2: Genetic map around the d-phenylserine dehydrogenase gene. orf1: putative transcriptional regulator gene, orf2: putative MFS transporter gene, orf3: l-phenylserine dehydrogenase gene, orf4: putative dihydroxy acid dehydratase gene, orf5: d-phenylserine dehydrogenase gene, orf6: putative ABC peptide transporter gene.

Mentions: To determine the nucleotide sequence of upstream and downstream regions of the gene encoding d-phenylserine dehydrogenase, inverse PCR was carried out. As a result, a 9,246-bp nucleotide sequence containing at least six open reading frames (ORFs) was determined (Figure 2). The transcriptional directions of orf1 and orf6 are opposite to those of the four other ORFs. Postulated promoter and terminator sequences are located immediately upstream of orf2 and downstream of d-phenylserine dehydrogenase encoding orf5, respectively. These observations suggest that orf2, orf3, orf4, and orf5 may form an operon.


Identification, Cloning, and Characterization of l-Phenylserine Dehydrogenase from Pseudomonas syringae NK-15.

Ueshima S, Muramatsu H, Nakajima T, Yamamoto H, Kato S, Misono H, Nagata S - Enzyme Res (2010)

Genetic map around the d-phenylserine dehydrogenase gene. orf1: putative transcriptional regulator gene, orf2: putative MFS transporter gene, orf3: l-phenylserine dehydrogenase gene, orf4: putative dihydroxy acid dehydratase gene, orf5: d-phenylserine dehydrogenase gene, orf6: putative ABC peptide transporter gene.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2963168&req=5

fig2: Genetic map around the d-phenylserine dehydrogenase gene. orf1: putative transcriptional regulator gene, orf2: putative MFS transporter gene, orf3: l-phenylserine dehydrogenase gene, orf4: putative dihydroxy acid dehydratase gene, orf5: d-phenylserine dehydrogenase gene, orf6: putative ABC peptide transporter gene.
Mentions: To determine the nucleotide sequence of upstream and downstream regions of the gene encoding d-phenylserine dehydrogenase, inverse PCR was carried out. As a result, a 9,246-bp nucleotide sequence containing at least six open reading frames (ORFs) was determined (Figure 2). The transcriptional directions of orf1 and orf6 are opposite to those of the four other ORFs. Postulated promoter and terminator sequences are located immediately upstream of orf2 and downstream of d-phenylserine dehydrogenase encoding orf5, respectively. These observations suggest that orf2, orf3, orf4, and orf5 may form an operon.

Bottom Line: The purified ORF3 enzyme showed l-phenylserine dehydrogenase activity.The genes encoding l-phenylserine dehydrogenase and d-phenylserine dehydrogenase, which is induced by phenylserine, are located in a single operon.The reaction products of both enzymatic reactions were 2-aminoacetophenone and CO(2).

View Article: PubMed Central - PubMed

Affiliation: The United Graduate School of Agricultural Science, Ehime University, 3-5-7 Tarumi, Matsuyama 790-8566, Japan.

ABSTRACT
The gene encoding d-phenylserine dehydrogenase from Pseudomonas syringae NK-15 was identified, and a 9,246-bp nucleotide sequence containing the gene was sequenced. Six ORFs were confirmed in the sequenced region, four of which were predicted to form an operon. A homology search of each ORF predicted that orf3 encoded l-phenylserine dehydrogenase. Hence, orf3 was cloned and overexpressed in Escherichia coli cells and recombinant ORF3 was purified to homogeneity and characterized. The purified ORF3 enzyme showed l-phenylserine dehydrogenase activity. The enzymological properties and primary structure of l-phenylserine dehydrogenase (ORF3) were quite different from those of d-phenylserine dehydrogenase previously reported. l-Phenylserine dehydrogenase catalyzed the NAD(+)-dependent oxidation of the β-hydroxyl group of l-β-phenylserine. l-Phenylserine and l-threo-(2-thienyl)serine were good substrates for l-phenylserine dehydrogenase. The genes encoding l-phenylserine dehydrogenase and d-phenylserine dehydrogenase, which is induced by phenylserine, are located in a single operon. The reaction products of both enzymatic reactions were 2-aminoacetophenone and CO(2).

No MeSH data available.


Related in: MedlinePlus