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

Abstract

Enzymes performing the initial reaction of aromatic amino acid biosynthesis, 2-keto-3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthases, exist as two distinct homology classes. The three classic Escherichia coli paralogs are AroA I proteins, but many members of the Bacteria possess the AroA II class of enzyme, sometimes in combination with AroA I proteins. AroA II 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 AroA II protein functions as the sole DAHP synthase supporting aromatic amino acid biosynthesis. X. campestris AroA II 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 AroA II 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 AroA II of higher plants, X. campestris AroA II was not hysteretically activated by dithiols. Compared to plant AroA II proteins, differences in divalent metal activation were also observed. Phylogenetic tree analysis shows that AroA II originated within the Bacteria domain, and it seems probable that higher-plant plastids acquired AroA II from a gram-negative bacterium via endosymbiosis. The X. campestris AroA II protein is suggested to exemplify a case of analog displacement whereby an ancestral aroA I species was discarded, with the aroA II replacement providing an alternative pattern of allosteric control. Three subgroups of AroA II 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.Microorganisms and plants engage 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (EC 4.1.2.15) as the initial catalyst for the commitment of phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) to the biosynthesis of aromatic amino acids, vitamin K, folic acid, and ubiquinone (4). The pathway contributes heavily to the biochemical individuality of organisms, with an enormous variety of peripheral branch connections known. Various pigments, siderophores, signaling compounds, defense metabolites, structural compounds, antibiotics, and other secondary metabolites are derived from this pathway (4).DAHP synthases fall into two distinct homology families. The AroA I family is divided into subfamilies AroA I␣ and AroA I␤ (32). The AroA I␣ subfamily is widely represented in gram-negative bacteria in which differentially regulated paralogs are commonly exp...

Genetic and biochemical identification of the chorismate mutase from Corynebacterium glutamicum

“…However, CM0819 and DS2098 from C. glutamicum differ from the bifunctional CM from B. subtilis in that the former was more sensitive to chorismate regulation (this study) and the latter was more sensitive to prephenate regulation (Jensen & Nester, 1965). Previously, Gosset et al (2001) reported that DAHP synthase from Xanthomonas campestris was subject to allosteric inhibition by chorismate and was slightly inhibited by Trp. We found that DS2098 alone was not subject to inhibition by chorismate (this study) and was slightly inhibited by Trp (Liu et al, 2008).…”

(De)regulation of key enzyme steps in the shikimate pathway and phenylalanine-specific pathway of the actinomycete Amycolatopsis methanolica

“…This type of DAHP synthase is often involved in the biosynthesis of secondary metabolites, such as the ansamycin antibiotic rifamycin in A. mediterranei (Yu et al, 2001), phenazine in P. aureofaciens (Pierson et al, 1995), aurachin in Stigmatella aurantiaca (Silakowski et al, 2000), chloramphenicol in Streptomyces venezuelae (He et al, 2001) and ansatrienin and naphthomycin in Streptomyces collinus (Chen et al, 1999). In contrast, in Xanthomonas campestris a plant-type enzyme functions as the sole DAHP synthase supporting aromatic amino acid biosynthesis (Gosset et al, 2001).…”

Tyrosine Latching of a Regulatory Gate Affords Allosteric Control of Aromatic Amino Acid Biosynthesis

“…Regulation of the catalytic activity of DAH7PS has been shown to be an important mechanism for control of cellular levels of aromatic compounds in microorganisms and plants (3). 13 C NMR studies using whole cells of Escherichia coli have demonstrated that feedback inhibition of DAH7PS is the main mechanism for controlling carbon flow into the shikimate pathway (4). Different organisms employ various strategies for this feedback inhibition.…”

“…This decoration of the barrel has been directly implicated in allosteric regulation of DAH7PS activity (8). DAH7PS enzymes have been divided into two types on the basis of protein size and amino acid sequence similarity (11): the smaller type I enzymes (30 -40 kDa), which can be further subdivided based on sequence into subtypes I␣ and I␤, and the larger type II enzymes (Ͼ50 kDa) (12,13). The sequence variability that defines the types and subtypes is directly paralleled in the structural variations that have been observed for DAH7PS enzymes (supplemental Fig.…”