CLONING AND CHARACTERIZATION OF A HETEROLOGOUSLY EXPRESSED BIFUNCTIONAL CHORISMATE SYNTHASE FLAVIN REDUCTASE FROM NEUROSPORA-CRASSA

Activities of all chorismate synthases (CS) so far analyzed are absolu tely dependent upon reduced flavin. For monofunctional CSs, which repr esent the only class of CSs that have yet been cloned, the flavin must be reduced either (photo-)chemically or by a separable flavin reducta se (FR) for in vitro activity. Neurospora crassa CS, in contrast, poss esses an intrinsic FR activity and represents the only firmly establis hed member of a bifunctional class of CSs. To better understand this b ifunctional protein, a cDNA from an N. crassa expression library encod ing a 46.4-kDa protein was cloned by complementation of the CS-deficie nt Escherichia coli strain AB2849. The deduced amino acid sequence was highly similar (79%) to a previously isolated Saccharomyces cerevisia e CS. The N. crassa sequence was unequivocally shown to encode the bif unctional CS/FR by analysis of the purified protein expressed in E. co il. Based on sequence comparisons with known monofunctional CSs, two r egions of 18 internal residues and 29 C-terminal residues unique to N. crassa CS were deleted, and the constructs were also expressed in E. coil. The presence of these regions was found not essential for comple mentation of the CS- phenotype of E. coil strain AB2849. Although a 3. 5-fold decline in specific activity of the purified CS from cells expr essing the C-terminal deletion construct was observed, bifunctional ac tivity was not eliminated. These data strongly suggest that the domain (s) responsible for reduction of flavin lie(s) within regions in which homology is also shared among monofunctional CSs.