The AroQ and PheA Domains of the Bifunctional P-Protein fromXanthomonas campestrisin a Context of Genomic Comparison

Abstract

The gene (denoted aroQ_p.pheA) encoding the bifunctional P-protein (chorismate mutaseP/prephenate dehydratase) from Xanthomonas campestris was cloned. aroQ_p.pheA is essential for L-phenylalanine biosynthesis. DNA sequencing of the smallest subclone capable of functional complementation of an Escherichia coli phenylalanine auxotroph revealed a putative open reading frame (ORF) of 1200 bp that would encode a 43,438-Da protein. AroQ_p.PheA exhibited 51% amino acid identity with a Pseudomonas stutzeri homologue and greater than 30% identities with AroQ_p.PheA proteins from Haemophilus influenzae, Neisseria gonorrhoeae, and a number of enteric bacteria. AroQ_p.PheA from X. campestris, when expressed in E. coli, possesses a 40-residue amino-terminal extension that is lysine-rich and that is absent in all of the AroQ_p.PheA homologues known at present. About 95% of AroQ_p.PheA was particulate and readily sedimented by low-speed centrifugation. Soluble preparations of cloned AroQ_p.PheA exhibited a native molecular mass of 81,000 Da, indicating that the active enzyme species is a homodimer. These preparations were unstable after purification of about 40-fold, even in the presence of glycerol, which was an effective protectant before fractionation. When AroQ_p.PheA was overproduced by a T₇ translation vector, unusual inclusion bodies having a macromolecular structure consisting of protein fibrils were observed by electron microscopy. Insoluble protein collected at low-speed centrifugation possessed high catalytic activity. The single band obtained via SDS-PAGE was used to confirm the translational start via N-terminal amino acid sequencing. A perspective on the evolutionary relationships of monofunctional AroQ and PheA proteins and the AroQ_p.PheA family of proteins is presented. A serC gene located immediately upstream of X. campestris aroQ_p.pheA appears to reflect a conserved gene organization, and both may belong to a single transcriptional unit. Rationale for terminology: It is unfortunate that the early Escherichia coli auxotrophs for phenylalanine and tyrosine were denoted pheA and tyrA before it was known that each gene product was bifunctional, carrying two catalytic domains. Each domain corresponds to individual proteins in many other or