Expression and purification of two isozymes of clavaminate synthase and initial characterization of the iron binding site. General error analysis in polymerase chain reaction amplification.
Clavaminate synthase is an Fe(2+)-, O2-, and alpha-ketoglutarate-dependent oxygenase that catalyzes three transformations in the biosynthesis of the important beta-lactamase inhibitor clavulanic acid. The genes from Streptomyces clavuligerus encoding two isoenzymes of clavaminate synthase have been over-expressed in Escherichia coli to give soluble proteins whose reactions, kinetic properties, and molecular masses are in excellent agreement with the wild-type isozymes. Preliminary investigation of the active site of clavaminate synthase was undertaken using diethyl pyrocarbonate and N-ethylmaleimide. Each was inhibitory to catalytic activity. Protection from inactivation in the presence of these reagents by Fe2+, O2, and alpha-ketoglutaric acid was thwarted by the rapid self-inactivation of the enzyme in the absence of substrate. However, protection was achieved when Co2+, a potent competitive inhibitor of clavaminate synthase 2 with respect to Fe2+, was substituted. This is consistent with the presence of histidine and cysteine, respectively, at or near the active site and possibly involved in iron binding. In the course of constructing the expression vector, a simply applied general error analysis of the polymerase chain reaction was formulated to calculate the proportion of correctly replicated DNA and guide the design of experiments using this method.
Busby
R W
RW
Department of Chemistry, Johns Hopkins University, Baltimore, Maryland 21218.
Chang
M D
MD
Busby
R C
RC
Wimp
J
J
Townsend
C A
CA
D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D005033:Ethylmaleimide / Q000494:pharmacology; D007501:Iron / Q000378:metabolism*; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010957:Plasmids; D016133:Polymerase Chain Reaction; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics
D004269:DNA, Bacterial; D007527:Isoenzymes; D007501:Iron; D006899:Mixed Function Oxygenases; C066046:clavaminate synthase; D004047:Diethyl Pyrocarbonate; D005033:Ethylmaleimide