Purification and Partial Characterization of the B Subunit ofSerratia marcescensTryptophan Synthetase

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Abstract
A trpE mutant of S. marcescens (E-7) was isolated, and the multimeric enzyme tryptophan synthetase (EC 4.2.1.20) was purified to homogeneity from derepressed cells. The A and B subunits were resolved, and the B subunit was partially characterized and compared with the Escherichia coli B subunit as part of a comparative evolution study of the trpB cistron of the trp operon in the Enterobacteriaceae. The S. marcescens B subunit is a dimer (.beta.2), and its MW was to be 89,000. The separate subunits (.beta. monomers) had MW of approximately 43,000. The B subunit required pyridoxal phosphate for catalytic activity and had an apparent Km of 9 .times. 10-6 M. The N [amino] terminus of the B subunit was unavailable for reaction with terminal amine reagents (blocked), but carboxypeptidase digestion released a C[carboxyl]-terminal isoleucine. Using S. marcescens B antiserum in agar immunodiffusion gave an almost complete reaction of identity between the B subunits of S. marcescens and E. coli. The antiserum was used in microcomplement fixation, allowing for a comparison of the overall antigenic surface structure of the 2 B subunits. The index of dissimilarity for the heterologous E. coli enzyme compared with the homologous S. marcescens enzyme was 2.4, indicating extensive similarity of the 2 proteins at their surfaces. Comparative antiserum neutralization of B-subunit enzyme activity showed the E. coli enzyme to cross-react 85% and the S. marcescens enzyme. The biochemical and immunochemical parameters used in this study showed that the S. marcescens and E. coli B subunits were either identical or very similar. Apparently, the trpB cistron of the trp operon is a relatively conserved gene in the Enterobacteriaceae.

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