In vitro analysis of the chain termination reaction in the synthesis of poly-(R)-beta-hydroxybutyrate by the class III synthase from Allochromatium vinosum.
Allochromatium vinosum polyhydroxyalkanoate synthase catalyzes formation of poly-(R)-3-hydroxybutyrate (PHB) from (R)-3-hydroxybutyryl-coenzyme A (HB-CoA). (R)-3-Hydroxybutyryl-N-acetylcysteamine (HB-NAC) is an alternative substrate for this synthase in vitro, with a turnover 1% that of HB-CoA. With HB-NAC, the molecular weight (M(w)) of PHB produced at substrate-to-enzyme ratios of 1500-15 000 is approximately 75 kDa. (1)H NMR shows that PHB produced has hydroxybutyrate at the alcohol end and N-acetylcysteamine (NAC) at the carboxylate end of the polymer. Exogenous NAC has no effect on the M(w) of PHB produced with HB-CoA or HB-NAC in vitro, whereas PHB from a polymerization reaction with both HB-CoA and HB-NAC has intermediate M(w). These results can be accommodated by two models. In the first, NAC liberated at the active site during polymerization acts as a chain transfer agent. In the second, there is a noncovalent polymer intermediate covalently linked to NAC, which can dissociate from the active site.
Lawrence
Adam G
AG
Department of Biology, Biomaterials Science & Engineering Laboratory, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Choi
Joonho
J
Rha
Chokyun
C
Stubbe
Joanne
J
Sinskey
Anthony J
AJ
D000217:Acyltransferases / Q000378:metabolism*; D002841:Chromatiaceae / Q000201:enzymology*; D004926:Escherichia coli / Q000378:metabolism; D006885:Hydroxybutyrates / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008970:Molecular Weight; D011091:Polyesters / Q000378:metabolism*; D000094122:Polyhydroxybutyrates
D000217:Acyltransferases; D006885:Hydroxybutyrates; D011091:Polyesters; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates; C060970:poly-beta-hydroxybutyrate polymerase