3-Hydroxy-3-methylglutaryl-CoA lyase: expression and isolation of the recombinant human enzyme and investigation of a mechanism for regulation of enzyme activity.
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.
Roberts
J R
JR
Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53226.
Narasimhan
C
C
Hruz
P W
PW
Mitchell
G A
GA
Miziorko
H M
HM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003545:Cysteine / Q000378:metabolism; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006801:Humans; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D007652:Oxo-Acid-Lyases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid
D011994:Recombinant Proteins; D004247:DNA; D007652:Oxo-Acid-Lyases; C021410:3-hydroxy-3-methylglutaryl-coenzyme A lyase; D003545:Cysteine