Avian 3-hydroxy-3-methylglutaryl-CoA synthase. Characterization of a recombinant cholesterogenic isozyme and demonstration of the requirement for a sulfhydryl functionality in formation of the acetyl-enzyme reaction intermediate.
cDNA encoding avian liver hydroxymethylglutaryl-CoA synthase has been cloned into a pET vector, and the resulting expression plasmid has been used to transform Escherichia coli BL21 (DE3). Heterologous expression of hydroxymethylglutaryl-CoA synthase occurs upon growth of this bacterial strain in the presence of isopropyl-1-thio-beta-D-galactopyranoside, with the target enzyme representing over 20% of total cellular protein. Recombinant enzyme is soluble and stable in crude E. coli extracts, facilitating its isolation in homogeneous form. With respect to specific activity, acylation stoichiometry, Km,Ac-CoA, and binding of a spin-labeled substrate analog, the recombinant enzyme is equivalent to avian enzyme, suggesting its utility for mechanistic and structural studies. Our earlier prediction that this avian cDNA encodes the cholesterogenic cytosolic isozyme is supported by a series of experimental observations. Upon SDS-polyacrylamide gel electrophoresis, the recombinant synthase exhibits mobility in agreement with the 57.6-kDa deduced molecular mass, which exceeds the 53-kDa estimate and experimental observation for the ketogenic mitochondrial isozyme. Activity of the recombinant synthase is stimulated by Mg2+, as predicted for the cholesterogenic cytosolic isozyme and in contrast to the inhibition observed for the mitochondrial isozyme. Although antibody prepared against avian mitochondrial synthase effectively detects both avian mitochondrial and recombinant synthases on Western blots, antibody prepared against rodent cytosolic synthase discriminates between the two proteins, sensitively detecting recombinant enzyme while reacting poorly with authentic mitochondrial enzyme. Directed mutagenesis of the recombinant synthase has been performed to produce a C129S variant, in which the sulfhydryl previously implicated in formation of the acetyl-S-enzyme reaction intermediate is replaced by a hydroxyl group. EPR measurements on the binary C129S-spin-labeled acyl-CoA complex demonstrate that the mutant's substrate binding site is unperturbed in comparison with wild-type protein. These data illustrate the utility of spin-labeled substrate analogs as tools to stringently evaluate the structural integrity of engineered proteins. C129S is catalytically inactive (10(5)-fold decrease in kcat) despite retaining the ability to form noncovalent complexes with acetyl-CoA or a spin-labeled acetyl-CoA analog. The demonstrated failure of C129S to form a covalent acyl-O-enzyme species accounts for these observations; data derived from experiments performed with a C129G mutant confirm this conclusion. These results distinguish hydroxymethylglutaryl-CoA synthase from beta-ketoacyl thiolase.(ABSTRACT TRUNCATED AT 400 WORDS)
Misra
I
I
Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53226.
Narasimhan
C
C
Miziorko
H M
HM
D000105:Acetyl Coenzyme A / Q000378:metabolism*; D000818:Animals; D001717:Birds; D002848:Chromatography, DEAE-Cellulose; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006904:Hydroxymethylglutaryl-CoA Synthase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D008930:Mitochondria, Liver / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D015183:Restriction Mapping; D013438:Sulfhydryl Compounds / Q000378:metabolism
D007527:Isoenzymes; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D000105:Acetyl Coenzyme A; D006904:Hydroxymethylglutaryl-CoA Synthase; D008274:Magnesium