Structure of Escherichia coli Aminodeoxychorismate Synthase:  Architectural Conservation and Diversity in Chorismate-Utilizing Enzymes^,

Abstract

Aminodeoxychorismate synthase is part of a heterodimeric complex that catalyzes the two-step biosynthesis of 4-amino-4-deoxychorismate, a precursor of p-aminobenzoate and folate in microorganisms. In the first step, a glutamine amidotransferase encoded by the pabA gene generates ammonia as a substrate that, along with chorismate, is used in the second step, catalyzed by aminodeoxychorismate synthase, the product of the pabB gene. Here we report the X-ray crystal structure of Escherichia coli PabB determined in two different crystal forms, each at 2.0 A resolution. The 453-residue monomeric PabB has a complex alpha/beta fold which is similar to that seen in the structures of homologous, oligomeric TrpE subunits of several anthranilate synthases of microbial origin. A comparison of the structures of these two classes of chorismate-utilizing enzymes provides a rationale for the differences in quaternary structures seen for these enzymes, and indicates that the weak or transient association of PabB with PabA during catalysis stems at least partly from a limited interface for protein interactions. Additional analyses of the structures enabled the tentative identification of the active site of PabB, which contains a number of residues implicated from previous biochemical and genetic studies to be essential for activity. Differences in the structures determined from phosphate- and formate-grown crystals, and the location of an adventitious formate ion, suggest that conformational changes in loop regions adjacent to the active site may be needed for catalysis. A surprising finding in the structure of PabB was the presence of a tryptophan molecule deeply embedded in a binding pocket that is analogous to the regulatory site in the TrpE subunits of the anthranilate synthases. The strongly bound ligand, which cannot be dissociated without denaturation of PabB, may play a structural role in the enzyme since there is no effect of tryptophan on the enzymic synthesis of aminodeoxychorismate. Extensive sequence similarity in the tryptophan-binding pocket among several other chorismate-utilizing enzymes, including isochorismate synthase, suggests that they too may bind tryptophan for structural integrity, and corroborates early ideas on the evolution of this interesting enzyme family.

The Structure of MbtI from Mycobacterium tuberculosis , the First Enzyme in the Biosynthesis of the Siderophore Mycobactin, Reveals It To Be a Salicylate Synthase

“…The pyruvate lyase enzymes PabC and PchB are themselves not related and are proposed to work via quite distinct mechanisms: the former is a pyridoxal phosphate-containing enzyme related to amino acid aminotransferases (24), while the latter shows weak chorismate mutase activity and is related to AroQ-type chorismate mutases (17). It has been suggested that the presence of Lys274 in PabB may both influence the specificity of the reaction and predict the need for a separate lyase (39). Arguing against this being so, PchA has an alanine residue in the equivalent position, in common with the other lyase-competent enzymes TrpE, MbtI, and Irp9.…”

“…However, His334 is on the mobile ␤16-␤17 loop and Thr271 on the adjacent ␤14 strand, so are both swung away from the active site in this "open" form; a ligand-binding role for these residues may be the major factor in loop closure. Adjacent to this part of the active site is Ala269, which is conserved as alanine in the Irp9 and TrpE enzymes but corresponds to Lys274 in PabB, where it has been proposed to discriminate between C-2 and C-4 substitution on chorismate substrates (39) and has recently been shown to be of critical importance for catalytic activity, as it forms a covalent intermediate during the reaction (3,4,27).…”

“…The enzymes that catalyze these conversions of chorismate share a degree of sequence identity (typically on the order of 20% on a pairwise basis) that implies related structures. Three-dimensional structures are currently available for salicylate synthase (Irp9) from Yersinia enterocolitica (29), for anthranilate synthase (TrpE) from Sulfolobus solfataricus, Serratia marcescens, and Salmonella enterica serovar Typhimurium (31,36,50), and for aminodeoxychorismate synthase (PabB) from Escherichia coli (39). Anthranilate synthase is a hetero-oligomeric complex composed of the products of the trpE and trpG genes.…”

“…In contrast, the salicylate synthase from Y. enterocolitica is homodimeric. Although the structures of TrpE, PabB, and Irp9 share a common fold (29,39), the fine structural differences that enable the production of different products by related enzymes (Fig. 2) are of great interest, particularly given the attractiveness of these enzymes for structure-based drug design.…”

Characterization of the Role of para- Aminobenzoic Acid Biosynthesis in Folate Production by Lactococcus lactis

“…In Escherichia coli, chorismate is converted via chorismate synthetase components I and II (PabB and PabA, EC 6.3.5.8) into 4-amino-4-deoxychorismate. Subsequently, pyruvate is cleaved by 4-amino-4-deoxychorismate lyase (PabC, EC 4.1.3.38), to result in pABA (10,26) (Fig. 1).…”