The aroQ and pheA domains of the bifunctional P-protein from Xanthomonas campestris in a context of genomic comparison.
The gene (denoted aroQp.pheA) encoding the bifunctional P-protein (chorismate mutase-P/prephenate dehydratase) from Xanthomonas campestris was cloned. aroQp.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. AroQp.PheA exhibited 51% amino acid identity with a Pseudomonas stutzeri homologoue and greater than 30% identities with AroQp.PheA proteins from Haemophilus influenzae, Neisseria gonorrhoeae, and a number of enteric bacteria. AroQp.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 AroQp.PheA homologues known at present. About 95% of AroQp.PheA was particulate and readily sedimented by low-speed centrifugation. Soluble preparations of cloned AroQp.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 AroQp.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 AroQp.PheA family of proteins is presented. A serC gene located immediately upstream of X. campestris aroQp.pheA appears to reflect a conserved gene organization, and both may belong to a single transcriptional unit.
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Department of Microbiology and Cell Science, University of Florida, Gainesville, USA.
Williams
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Aldrich
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Xie
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Gabriel
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Jensen
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D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D001483:Base Sequence; D002826:Chorismate Mutase / Q000235:genetics* / Q000378:metabolism; D003429:Cross Reactions; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D002479:Inclusion Bodies / Q000648:ultrastructure; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny; D011302:Prephenate Dehydratase / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D016959:Xanthomonas campestris / Q000235:genetics* / Q000648:ultrastructure
D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; C071216:P-protein, E coli; D011302:Prephenate Dehydratase; D002826:Chorismate Mutase