Cloning, sequencing, and expression of Rhodococcus L-phenylalanine dehydrogenase. Sequence comparisons to amino-acid dehydrogenases.
L-Phenylalanine dehydrogenase catalyzes the NAD(+)-dependent, reversible, oxidative deamination of L-phenylalanine to form ammonia, phenyl pyruvate, and NADH. The enzyme has been purified to homogeneity from Rhodococcus sp. M4, and a partial amino acid sequence was obtained. A cosmid library of Rhodococcus sp. M4 genomic DNA was prepared and used to isolate a 2.5-kilobase PstI fragment that contained the pdh gene. The open reading frame of 1068 nucleotides encodes a polypeptide of 356 amino acids, portions of which match the amino acid sequence determined for the purified enzyme. Expression of the Rhodococcus pdh gene in Escherichia coli, which does not contain a phenylalanine dehydrogenase activity, yields a soluble enzyme exhibiting phenylalanine dehydrogenase activity. Both the enzyme purified from Rhodococcus and the enzyme expressed in E. coli are post-translationally modified by removal of the amino-terminal methionine. The overall amino acid sequence is homologous to previously reported sequences of leucine and phenylalanine dehydrogenases as well as several glutamate dehydrogenases. The amino-terminal portion of the enzyme contains residues involved in L-amino acid binding and catalysis, while the carboxyl-terminal portion contains the presumptive dinucleotide-binding domain. A detailed sequence comparison of Rhodococcus phenylalanine dehydrogenase with leucine, phenylalanine, and glutamate dehydrogenases suggests residues involved in general amino acid binding and others that provide for amino acid discrimination.
Brunhuber
N M
NM
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461.
Banerjee
A
A
Jacobs
W R
WR
Blanchard
J S
JS
D000594:Amino Acid Oxidoreductases / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001483:Base Sequence; D005075:Biological Evolution; D003001:Cloning, Molecular; D005798:Genes, Bacterial / Q000235:genetics*; D015698:Genomic Library; D001411:Geobacillus stearothermophilus / Q000201:enzymology / Q000235:genetics; D050803:Leucine Dehydrogenase; D008969:Molecular Sequence Data; D012240:Rhodococcus / Q000201:enzymology / Q000235:genetics*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D000596:Amino Acids; D000594:Amino Acid Oxidoreductases; D050803:Leucine Dehydrogenase; C022342:phenylalanine oxidase