3-Hydroxy-3-methylglutaryl-CoA lyase: expression and isolation of the recombinant human enzyme and investigation of a mechanism for regulation of enzyme activity

J Biol Chem. 1994 Jul 8;269(27):17841-6.


cDNA encoding the mature form of human hydroxy-methylglutaryl-CoA (HMG-CoA) lyase, a mitochondrial matrix protein, has been used to prepare expression plasmids appropriate for production of this protein in Escherichia coli. Using a T7 RNA polymerase-based pET system, HMG-CoA lyase was overexpressed but largely recovered in an insoluble, catalytically inactive form. In contrast, an expression plasmid (pTrcHL-1), derived from pTrc99a, supported production of soluble, active enzyme. A synthetic oligonucleotide cassette was employed to produce an enzyme variant in which cysteine was replaced by serine at position 323. Both wild-type and C323S HMG-CoA lyases were isolated in homogeneous form and characterized. The function of Cys-323 in influencing catalytic activity in vitro has been investigated by comparing the response of wild-type and C323S lyases to oxidation and reduction. Additionally, the consequences of treatment of these enzymes with the sulfhydryl-directed bifunctional reagent, o-phenylenedimaleimide have been determined. The results support the hypothesis that a thiol/disulfide exchange mechanism affects enzyme activity in vitro and indicate that Cys-323 residues on adjacent subunits of the homodimeric native enzyme are suitably positioned to form an intersubunit cross-link upon oxidative inactivation and disulfide formation.

Publication types

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

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Cloning, Molecular
  • Cysteine / metabolism
  • DNA
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli
  • Humans
  • Molecular Sequence Data
  • Oxidation-Reduction
  • Oxo-Acid-Lyases / genetics
  • Oxo-Acid-Lyases / isolation & purification
  • Oxo-Acid-Lyases / metabolism*
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / isolation & purification
  • Sequence Homology, Amino Acid


  • Recombinant Proteins
  • DNA
  • Oxo-Acid-Lyases
  • 3-hydroxy-3-methylglutaryl-coenzyme A lyase
  • Cysteine