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

Enzymes performing the initial reaction of aromatic amino acid biosynthesis , 2-keto-3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) syntheses, exis t as two distinct homology classes. The three classic Escherichia coil para logs are AroA(I) proteins, but many members of the Bacteria possess the Aro A(II) class of enzyme, sometimes in combination with AroA(I) proteins. AroA (II) DAHP syntheses until now have been shown to be specifically dedicated to secondary metabolism (e.g., formation of ansamycin antibiotics or phenaz ine pigment). In contrast, here we show that the Xanthomonas campestris Aro A(II) protein functions as the sole DAHP synthase supporting aromatic amino acid biosynthesis. X. campestris AroA(II) was cloned in E. coil by functio nal complementation, and genes corresponding to two possible translation st arts were expressed. We developed a 1-day partial purification method (> 99 %) for the unstable protein. The recombinant AroA(II) protein was found to be subject to an allosteric pattern of sequential feedback inhibition in wh ich 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 AroA(II) of higher plants, X. c ampestris AroA(II) was not hysteretically activated by dithiols. Compared t o plant AroA(II) proteins, differences in divalent metal activation were al so observed. Phylogenetic tree analysis shows that AroA(II) originated with in the Bacteria domain, and it seems probable that higher-plant plastids ac quired AroA(II) from a gram-negative bacterium via endosymbiosis. The X. ca mpestris AroA(II) protein is suggested to exemplify a case of analog displa cement whereby an ancestral aroA(I) species was discarded, with the aroA(II ) replacement providing an alternative pattern of allosteric control. Three subgroups of AroA,, proteins can be recognized: a large, central group con taining the plant enzymes and that from X. campestris, one defined by a thr ee-residue deletion near the conserved KPRS motif, and one possessing a lar ger deletion further downstream.