Microbial Origin of Plant-Type 2-Keto-3-Deoxy-d-arabino-Heptulosonate 7-Phosphate Synthases, Exemplified by the Chorismate- and Tryptophan-Regulated Enzyme from Xanthomonas campestris

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American Society for Microbiology

RESUMO

Enzymes performing the initial reaction of aromatic amino acid biosynthesis, 2-keto-3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthases, exist as two distinct homology classes. The three classic Escherichia coli paralogs are AroAI proteins, but many members of the Bacteria possess the AroAII class of enzyme, sometimes in combination with AroAI proteins. AroAII DAHP synthases until now have been shown to be specifically dedicated to secondary metabolism (e.g., formation of ansamycin antibiotics or phenazine pigment). In contrast, here we show that the Xanthomonas campestris AroAII protein functions as the sole DAHP synthase supporting aromatic amino acid biosynthesis. X. campestris AroAII was cloned in E. coli by functional complementation, and genes corresponding to two possible translation starts were expressed. We developed a 1-day partial purification method (>99%) for the unstable protein. The recombinant AroAII protein was found to be subject to an allosteric pattern of sequential feedback inhibition in which chorismate is the prime allosteric effector. l-Tryptophan was found to be a minor feedback inhibitor. An N-terminal region of 111 amino acids may be located in the periplasm since a probable inner membrane-spanning region is predicted. Unlike chloroplast-localized AroAII of higher plants, X. campestris AroAII was not hysteretically activated by dithiols. Compared to plant AroAII proteins, differences in divalent metal activation were also observed. Phylogenetic tree analysis shows that AroAII originated within the Bacteria domain, and it seems probable that higher-plant plastids acquired AroAII from a gram-negative bacterium via endosymbiosis. The X. campestris AroAII protein is suggested to exemplify a case of analog displacement whereby an ancestral aroAI species was discarded, with the aroAII replacement providing an alternative pattern of allosteric control. Three subgroups of AroAII proteins can be recognized: a large, central group containing the plant enzymes and that from X. campestris, one defined by a three-residue deletion near the conserved KPRS motif, and one possessing a larger deletion further downstream.

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