X-ray crystal structures of HMG-CoA synthase from Enterococcus faecalis and a complex with its second substrate/inhibitor acetoacetyl-CoA.
Biosynthesis of the isoprenoid precursor, isopentenyl diphosphate, is a critical function in all independently living organisms. There are two major pathways for this synthesis, the non-mevalonate pathway found in most eubacteria and the mevalonate pathway found in animal cells and a number of pathogenic bacteria. An early step in this pathway is the condensation of acetyl-CoA and acetoacetyl-CoA into HMG-CoA, catalyzed by the enzyme HMG-CoA synthase. To explore the possibility of a small molecule inhibitor of the enzyme functioning as a non-cell wall antibiotic, the structure of HMG-CoA synthase from Enterococcus faecalis (MVAS) was determined by selenomethionine MAD phasing to 2.4 A and the enzyme complexed with its second substrate, acetoacetyl-CoA, to 1.9 A. These structures show that HMG-CoA synthase from Enterococcus is a member of the family of thiolase fold enzymes and, while similar to the recently published HMG-CoA synthase structures from Staphylococcus aureus, exhibit significant differences in the structure of the C-terminal domain. The acetoacetyl-CoA binary structure demonstrates reduced coenzyme A and acetoacetate covalently bound to the active site cysteine through a thioester bond. This is consistent with the kinetics of the reaction that have shown acetoacetyl-CoA to be a potent inhibitor of the overall reaction, and provides a starting point in the search for a small molecule inhibitor.
Steussy
C Nicklaus
CN
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907, USA.
Vartia
Anthony A
AA
Burgner
John W
JW
Sutherlin
Autumn
A
Rodwell
Victor W
VW
Stauffacher
Cynthia V
CV
D000090:Acetoacetates / Q000737:chemistry; D000214:Acyl Coenzyme A / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray / Q000379:methods; D003545:Cysteine / Q000737:chemistry; D013293:Enterococcus faecalis / Q000201:enzymology*; D006904:Hydroxymethylglutaryl-CoA Synthase / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D012645:Selenomethionine / Q000737:chemistry; D016415:Sequence Alignment; D013379:Substrate Specificity
D000090:Acetoacetates; D000214:Acyl Coenzyme A; C010667:acetoacetyl CoA; D012645:Selenomethionine; D006904:Hydroxymethylglutaryl-CoA Synthase; D003545:Cysteine