The Archaeoglobus Fulgidus D-lactate Dehydrogenase Is a Zn(2+) Flavoprotein

J Bacteriol. 1999 Dec;181(24):7580-7.

Abstract

Archaeoglobus fulgidus, a hyperthermophilic, archaeal sulfate reducer, is one of the few organisms that can utilize D-lactate as a sole source for both carbon and electrons. The A. fulgidus open reading frame, AF0394, which is predicted to encode a D-(-)-lactate dehydrogenase (Dld), was cloned, and its product was expressed in Escherichia coli as a fusion with the maltose binding protein (MBP). The 90-kDa MBP-Dld fusion protein was more efficiently expressed in E. coli when coexpressed with the E. coli dnaY gene, encoding the arginyl tRNA for the codons AGA and AGG. When cleaved from the fusion protein by treatment with factor Xa, the recombinant Dld (rDld) has an apparent molecular mass of 50 kDa, similar to that of the native A. fulgidus Dld enzyme. Both the purified MBP-Dld fusion protein and its rDld cleavage fragment have lactate dehydrogenase activities specific for D-lactate, are stable at 80 degrees C, and retain activity after exposure to oxygen. The flavin cofactor FAD, which binds rDld apoprotein with a 1:1 stoichiometry, is essential for activity.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • ATP-Binding Cassette Transporters*
  • Archaeoglobus fulgidus / enzymology*
  • Archaeoglobus fulgidus / genetics
  • Carrier Proteins / genetics
  • Cloning, Molecular
  • Escherichia coli
  • Escherichia coli Proteins*
  • Kinetics
  • L-Lactate Dehydrogenase / genetics
  • L-Lactate Dehydrogenase / metabolism*
  • Maltose-Binding Proteins
  • Methylphenazonium Methosulfate / metabolism
  • Monosaccharide Transport Proteins*
  • Open Reading Frames
  • Recombinant Fusion Proteins / metabolism
  • Zinc / metabolism*

Substances

  • ATP-Binding Cassette Transporters
  • Carrier Proteins
  • Escherichia coli Proteins
  • Maltose-Binding Proteins
  • Monosaccharide Transport Proteins
  • Recombinant Fusion Proteins
  • maltose transport system, E coli
  • Methylphenazonium Methosulfate
  • L-Lactate Dehydrogenase
  • Zinc