Selection and characterization of a mutant of the cloned gene for mandelate racemase that confers resistance to an affinity label by greatly enhanced production of enzyme

Abstract

The plasmid pSCR1 containing the gene for mandelate racemase (EC 5.1.2.2) from Pseudomonas putida (ATCC 12633) allows Pseudomonas aeruginosa (ATCC 15692) to grow on (R)-mandelate as its sole carbon source [Ransom, S. C., Gerlt, J. A., Powers, V. M., & Kenyon, G. L. (1988) Biochemistry 27, 540]; the chromosome of the P. aeruginosa host apparently does not contain the gene for mandelate racemase but does contain genes for the remaining enzymes in the mandelate pathway and enables growth on (S)-mandelate as carbon source. However, in the presence of alpha-phenylglycidate, an active-site-directed irreversible inhibitor (affinity label) of mandelate racemase, P. aeruginosa transformed with pSCR1 can utilize (S)-mandelate but not (R)-mandelate as carbon source. This inhibition of growth on (R)-mandelate provides a metabolic selection for mutants that are resistant to alpha-phenylglycidate. When (R)-mandelate is used as carbon source and alpha-phenylglycidate is present, a few colonies of P. aeruginosa transformed with pSCR1 grow slowly and appear on plates after several days. The plasmid isolated from these cells confers resistance to alpha-phenylglycidate on newly transformed cells of P. aeruginosa. This resistance to the affinity label is not due to a mutation within the primary structure of the enzyme. A single base change (C----A) located 87 bp upstream of the initiation codon for the gene for mandelate racemase was detected in three independent isolates of alpha-phenylglycidate-resistant colonies and appears responsible for a 30-fold increase in the amount of mandelate racemase encoded by the gene contained in the plasmid.(ABSTRACT TRUNCATED AT 250 WORDS)

The Substrate Spectrum of Mandelate Racemase: Minimum Structural Requirements for Substrates and Substrate Model

“…[11,12] The inducible octameric enzyme (subunit 39 kDa) has been made available in large amounts by fermentation of Pseudomonas putida ATCC 12633 using rac-mandelate as inducer [13] and it has been cloned. [14] Enzyme immobilization onto the cationic carrier DEAE-cellulose leads to enhanced activity and facilitates recovery. [15] Simple assays for mandelate-racemase activity based on circular dichroism [16] or on the decline of optical rotation over time [17] have been developed.…”

“…Although first hints on the relaxed substrate specificity of mandelate racemase were published quite early, [14] it was the availability of this enzyme by fermentation [13] that facilitated the exploration of its substrate tolerance considerably. [15,28] Moreover, molecular modeling showed that the hydrophobic binding pocket within the active site (which accommodates the phenyl moiety of the natural substrate, mandelate, see Fig.…”

“…While some reduction in activity is observed with m-substituents (4), o-substituted (5) and m, p-disubstituted mandelate derivatives (9, 10) are not accepted, presumably due to steric hindrance, which is most striking for the o-derivative. Total replacement of the phenyl moiety by a bulky naphthyl group (11) or by a heterocycle, such as furan (13,14) or thiophene (12) gave moderate to good racemization rates.…”

Glutamate racemization and catabolism inFusobacterium varium

“…The utilization of d ‐glutamate via a glutamate racemase expands the pool of possible energy substrates and provides F. varium with an environmental advantage. Other bacterial pathways starting with racemization as the first step are utilized for catabolism of ornithine [45] and mandelic acid [46], but racemases are present more commonly to provide d ‐amino acids as building blocks for bacterial cell walls [47]. While the formation of a small amount of d ‐glutamate in an assay of glutamate mutase from C. tetanomorphum was attributed to a glutamate racemase contamination [29,36], a later study reported that no glutamate racemase activity was detected in Clostridium species [48].…”

Identification and Characterization of a Mandelamide Hydrolase and an NAD(P)+-Dependent Benzaldehyde Dehydrogenase fromPseudomonas putidaATCC 12633

“…1). Genetic experiments indicated that the genes for the mandelate pathway were all closely clustered on the chromosome (36,38), and, subsequently, it was reported that the mandelate pathway genes for P. putida ATCC 12633 resided on a single 10.5-kb restriction fragment (35). Sequencing of part of that fragment showed that three of the genes-those encoding mandelate racemase (mdlA), Smandelate dehydrogenase (mdlB), and benzoylformate decarboxylase (mdlC)-were arranged in an operon (31,35).…”