Journal of Bacteriology, July 2001, p. 4061-4070, Vol. 183, No. 13
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.13.4061-4070.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Departamento de Microbiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos 62250, Mexico1; Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida 326112; Department of Chemistry, City College of New York, New York, New York 100313; and BioScience Division, Los Alamos National Laboratory, Los Alamos, New Mexico 875444
Received 31 October 2000/Accepted 3 April 2001
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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