Two Forms of NAD-Dependent d -Mandelate Dehydrogenase in Enterococcus faecalis IAM 10071

Abstract

Two forms of NAD-dependent d-mandelate dehydrogenase (d-ManDHs) were purified from Enterococcus faecalis IAM 10071. While these two enzymes consistently exhibited high activity toward large 2-ketoacid substrates that were branched at the C3 or C4 position, they gave distinctly different Km and V max values for these substrates and had distinct molecular weights by gel electrophoresis and gel filtration.

A New Family ofD-2-Hydroxyacid Dehydrogenases That ComprisesD-Mandelate Dehydrogenases and 2-Ketopantoate Reductases

“…The recombinant enzyme was purified by essentially the same purification procedure as that used for native D-ManDH2, and it exhibited an apparent molecular weight of 40 kDa on SDS-PAGE ( Fig. 2A), in good agreement with that of native D-ManDH2, 28) although somewhat larger than the molecular weight (34,331 Da) calculated from the deduced primary structure of the enzyme (see Fig. 3A) for unknown reasons.…”

“…3A) for unknown reasons. 28) the recombinant enzyme showed V m values for most substrates similar to those of DManDH2, rather than D-ManDH1. Together with the apparent molecular weight (Fig.…”

“…When cell growth reached an optical density of approximately 1.0 at 660 nm, the cultivation temperature was reduced to 25 C, and IPTG (final 1.0 mM) was added to the culture medium. The cells were harvested after additional cultivation at 25 C for 2 h. Purification of the recombinant enzymes expressed by pET-DmanDH, and pBK-DmanDH was performed according to the procedure for native DManDH2, 28) which involves ammonium sulfate precipitation, and Butyl Toyopearl, DEAE-Sepharose, and Resource Q (GE Healthcare Bio-Sciences) or Uno-Q (Bio-Rad Laboratories) column chromatography. The purity of the enzyme preparations was examined by SDS-PAGE following to Laemmli.…”

“…Nevertheless, their structural relation to the D-2-HydDH family remains uncertain, because little is known about the protein structure of D-ManDHs. 28 …”

“…[21][22][23][24] In spite of the great variety of their catalytic functions, however, there is no reported enzyme in the D-2-HydDH family that exhibits high catalytic activity toward C3-branched substrates. 17,18,22,25) Enzymes purified from Lactobacillus curvatus, 26) Enterococcus faecalis, 27,28) and also the yeast Rhodotorula graminis 29) are known to catalyze efficiently the conversion between benzoylformate (C 6 H 5 -CO-COO À ) and D-mandelate (C 6 H 5 -CHOH-COO À ), which possess a branched side chain at the C3 position, and are called D-mandelate dehydrogenases (D-ManDHs). Nevertheless, their structural relation to the D-2-HydDH family remains uncertain, because little is known about the protein structure of D-ManDHs.…”