Mandelate pathway of Pseudomonas putida: sequence relationships involving mandelate racemase, (S)-mandelate dehydrogenase, and benzoylformate decarboxylase and expression of benzoylformate decarboxylase in Escherichia coli.
The genes that encode the five known enzymes of the mandelate pathway of Pseudomonas putida (ATCC 12633), mandelate racemase (mdlA), (S)-mandelate dehydrogenase (mdlB), benzoylformate decarboxylase (mdlC), NAD(+)-dependent benzaldehyde dehydrogenase (mdlD), and NADP(+)-dependent benzaldehyde dehydrogenase (mdlE), have been cloned. The genes for (S)-mandelate dehydrogenase and benzoylformate decarboxylase have been sequenced; these genes and that for mandelate racemase [Ransom, S. C., Gerlt, J. A., Powers, V. M., & Kenyon, G. L. (1988) Biochemistry 27, 540] are organized in an operon (mdlCBA). Mandelate racemase has regions of sequence similarity to muconate lactonizing enzymes I and II from P. putida. (S)-Mandelate dehydrogenase is predicted to be 393 amino acids in length and to have a molecular weight of 43,352; it has regions of sequence similarity to glycolate oxidase from spinach and ferricytochrome b2 lactate dehydrogenase from yeast. Benzoylformate decarboxylase is predicted to be 499 amino acids in length and to have a molecular weight of 53,621; it has regions of sequence similarity to enzymes that decarboxylate pyruvate with thiamin pyrophosphate as cofactor. These observations support the hypothesis that the mandelate pathway evolved by recruitment of enzymes from preexisting metabolic pathways. The gene for benzoylformate decarboxylase has been expressed in Escherichia coli with the trc promoter, and homogeneous enzyme has been isolated from induced cells.
Tsou
A Y
AY
Department of Chemistry and Biochemistry, University of Maryland, College Park 20742.
Ransom
S C
SC
Gerlt
J A
JA
Buechter
D D
DD
Babbitt
P C
PC
Kenyon
G L
GL
D000095:Acetolactate Synthase / Q000235:genetics; D000429:Alcohol Oxidoreductases / Q000235:genetics; D000445:Aldehyde Oxidoreductases / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D002262:Carboxy-Lyases / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism; D005798:Genes, Bacterial; D019753:Intramolecular Lyases; D007535:Isomerases / Q000235:genetics; D007770:L-Lactate Dehydrogenase / Q000235:genetics; D008333:Mandelic Acids / Q000378:metabolism*; D008969:Molecular Sequence Data; D009876:Operon; D011549:Pseudomonas / Q000235:genetics* / Q000378:metabolism; D011767:Pyruvate Decarboxylase / Q000235:genetics; D011771:Pyruvate Oxidase / Q000235:genetics; D019998:Racemases and Epimerases / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid
D001426:Bacterial Proteins; D008333:Mandelic Acids; D011993:Recombinant Fusion Proteins; D000429:Alcohol Oxidoreductases; C020819:glycolic acid dehydrogenase; D007770:L-Lactate Dehydrogenase; D000445:Aldehyde Oxidoreductases; C027015:benzaldehyde dehydrogenase (NAD+); D011771:Pyruvate Oxidase; D000095:Acetolactate Synthase; D002262:Carboxy-Lyases; D011767:Pyruvate Decarboxylase; C019778:benzoylformate decarboxylase; D007535:Isomerases; D019998:Racemases and Epimerases; C021540:mandelate racemase; D019753:Intramolecular Lyases; C021811:muconate cycloisomerase; C037938:mandelic acid