Mandelate pathway of Pseudomonas putida: sequence relationships involving mandelate racemase, (S)-mandelate dehydrogenase, and benzoylformate decarboxylase and expression of benzoylformate decarboxylase in Escherichia coli

Abstract

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.

Strategic manipulation of the substrate specificity of Saccharomyces cerevisiae flavocytochrome b2

“…The flavin-binding domain of flavocytochrome b2 is closely related to several other 2-hydroxy acid dehydrogenases with different substrate specificities. These include the glycollate oxidase from spinach [18], lactate oxidase from Mycobacterium smegmatis [19], lactate dehydrogenase from E. coli [20], mandelate dehydrogenase from Pseudomonas putida [21] and long-chain hydroxy acid dehydrogenase from rat kidney [22]. The catalytically important residues are well conserved throughout this family but we have sought to determine the structural basis for substrate selectivity.…”

Purification and crystallization of benzoylformate decarboxylase

“…Previous isolations of the enzyme from P. putida resulted in specific activities of 193 U/mg (Hegeman, 1970) and 16.8 U/mg (Wilcocks et al, 1992); benzoylformate decarboxylase, with a specific activity of 46 U/mg, has also been isolated from Escherichia coli expressing the mdlC gene (Tsou et al, 1990). Our purification is based on the latter method.…”

“…Benzoylformate decarboxylase was expressed in E. coli containing the plasmid pBFDtrc as described previously (Tsou et al, 1990), except that the cells were induced with isopropyl-0-thiogalactopyranoside when they reached an OD of 1.2 (rather than 1 .O), and the wet cells were stored at -80 "C (rather than -20 "C). Increasing the scale of the fermentation from 9 L to 60 L resulted in comparable yields.…”

Mandelate Racemase and Mandelate Dehydrogenase Coexpressed RecombinantEscherichia coliin the Synthesis of Benzoylformate

“…To obtain the genes encoding racemase mdlA and mandelate dehydrogenase mldB from P. aeruginosa NUST, primers P1 and P2 were designed using the genomic sequence of Pseudomonas putida as target. 25) P1 with NdeI restriction site insertion and P2 with NotI restriction site insertion were as follows: P1, 5-CCCCATATGAGCCAGAATCTC-TTTAAC-3 and P2, 5-GCGGCCGCTTACACCAGATATTTCCCGA-3. The DNA fragment was amplified by PCR from the genome of P. aeruginosa NUST using P1 and P2.…”