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

Abstract

The enzymes of the mandelate metabolic pathway permit Pseudomonas putida ATCC 12633 to utilize either or both enantiomers of mandelate as the sole carbon source. The genes encoding the mandelate pathway were found to lie on a single 10.5-kb restriction fragment. Part of that fragment was shown to contain the genes coding for mandelate racemase, mandelate dehydrogenase, and benzoylformate decarboxylase arranged in an operon. Here we report the sequencing of the remainder of the restriction fragment, which revealed three further open reading frames, denoted mdlX, mdlY, and mdlD. All were transcribed in the opposite direction from the genes of the mdlABC operon. Sequence alignments suggested that the open reading frames encoded a regulatory protein (mdlX), a member of the amidase signature family (mdlY), and an NAD(P)؉ -dependent dehydrogenase (mdlD). The mdlY and mdlD genes were isolated and expressed in Escherichia coli, and the purified gene products were characterized as a mandelamide hydrolase and an NAD(P) ؉ -dependent benzaldehyde dehydrogenase, respectively.

Cofactor Recycling Using a Thermostable NADH Oxidase

“…Enzyme activity was inhibited by sulfhydryl agents such as Hg 2+ (23%), p-CMBA (0%), iodoacetamide (0%), and Nmethylmaleimide (0%), which indicates that a thiol group plays an important role in oxidation. PADH was not stimulated by potassium ions, unlike ALDH2 from S. cerevisiae and BADH from Pseudomonas putida (McLeish et al, 2003). PADH activity was inhibited by a high concentration (0.1 mM) of phenylacetaldehyde.…”

A Type I/Type III Polyketide Synthase Hybrid Biosynthetic Pathway for the Structurally Unique ansa Compound Kendomycin

“…Biosynthesis of the kendomycin starter unit compared to 3,5-DHPG biosynthesis in A. mediterranei [22] and the mandelate degradation pathway from P. putida. [32,33] The first two steps from the type III PKS-initiated kendomycin starter unit biosynthesis are identical to the 3,5-DHPG assembly process and the involved proteins from both pathways (Ken2-4,7/DpgA-D) show a high degree of similarity to each other. The produced 3,5-DHPGO, which is further transaminated in the 3,5-DHPG pathway is degraded to 3,5-DHBA by Ken5 and Ken6, which perform similar reactions as their homologues (MdlC and MdlD) from the mandelate degradation pathway.…”

“…[33,36] The subsequent oxidation of the al-A C H T U N G T R E N N U N G dyhyde group by the NAD-dependent benzaldehyde dehydrogenase (MdlD) leads to the formation of benzoic acid. [32] In analogy to these reaction steps, we propose that the MdlC homologue Ken5 transforms 3,5-DHPGO into 3,5-dihydroxybenzaldehyde (3,5-DHBAL), which is subsequently oxidized by the MdlD homologue Ken6 to the corresponding acid (3,5-dihydroxybenzoic acid, 3,5-DHBA; see Scheme 1). Because two additional biosynthetic genes (ken9 and ken10) are located downstream of ken7, we assume that the starter unit is further modified before entering the type I PKS assembly process.…”

“…Both enzymes are involved in the mandelate degradation pathway, which enables pseudomonads to use mandelate as a sole carbon source. [32,33] In four A C H T U N G T R E N N U N G reaction steps the compound (R)-mandelate is converted into benzoic acid (see Scheme 1), which can subsequently be metabolized through the b-ketoadipate pathway and the tricarboxylic acid cycle. The well-characterized benzoylformate decarboxylase (MdlC) from Pseudomonas putida ATCC 12 633 is a thiamine-diphosphate-dependent enzyme, which catalyzes the nonoxidative conversion of benzoylformate into benzaldehyde and carbon dioxide.…”

“…The corresponding proteins show homology to a benzoylformate decarboxylase and a benzaldehyde dehydrogenase from the mandelate degradation pathway of different Pseudomonas strains. [32,33] Further downstream, two putative modifying enzymes, a methyl transferase (Ken9), as well as a pyrroloquinoline quinone (PQQ)-dependent protein (Ken10) are encoded. A regulatory gene (ken11) is located directly after the putative ken9/10 operon.…”