Regulation of Aromatic Amino Acid Biosynthesis in Bacillus subtilis 168: I. EVIDENCE FOR AND CHARACTERIZATION OF A TRIFUNCTIONAL ENZYME COMPLEX

Abstract

Abstract A bifunctional enzyme complex consisting of 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) synthetase and chorismate mutase in strain 168 of Bacillus subtilis has been purified to near homogeneity by conventional purification procedures. A major protein band with two minor components was observed when the final preparation was subjected to gel electrophoresis at pH 9.3 and 7.0. DAHP synthetase and chorismate mutase activity were associated only with the major band. The significant property of the bifunctional enzyme complex is its relationship to shikimate kinase, another enzyme of the biosynthetic pathway. When cell-free preparations are chromatographed on Sephadex G-200, only a trace of shikimate kinase activity is recovered. When the chorismate mutase-DAHP synthetase complex fraction is mixed with fractions which show only traces of shikimate kinase activity, full catalytic activity of shikimate kinase is restored. The activity of the reconstructed shikimate kinase activity resembles the activity observed in crude extracts in several parameters. Single site mutants which lack chorismate mutase activity also lack DAHP synthetase and shikimate kinase activities. The close association of these three enzymes in vitro as well as the isolation of such pleiotrophic mutants strongly suggests that these three enzymes are part of a multi-enzyme complex in vivo. The present data are consistent with the hypothesis that this complex functions as a unit of feedback control.