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Crystal structure of the Escherichia coli shikimate kinase I (AroK) that confers sensitivity to mecillinam

Introduction. Escherichia coli shikimate kinase I (SKI, AroK; EC 2.7.1.71; 173 residues; predicted molecular mass 19,538 Da) catalyzes the fifth step in the sevenstep chorismate biosynthesis pathway unique to microorganisms, plants, and parasites. AroK is a member of the nucleoside monophosphate (NMP) kinase family and has been shown to convert shikimic acid to shikimate-3phosphate using adenosine triphosphate (ATP) as a cosubstrate. In E. coli, phosphorylation of shikimic acid is carried out by two isozymes: shikimate kinase I encoded by the aroK gene and shikimate kinase II (SKII, AroL) encoded by the aroL gene. Both genes are located in different parts of the bacterial chromosome and form parts of distinct operons. Expression of the aroK gene, unlike that of aroL (controlled by the TyrR and TrpR repressors), appears to be constitutive and the enzyme has approximately 100-fold lower affinity for shikimic acid than SKII with an estimated Km of 20 mM for AroK and 200 M for SKII. AroK can functionally substitute for SKII, but double mutants in aroK and aroL are aromatic amino acid auxotrophs. These observations suggest that SKII is the dominant enzyme of the pathway, with AroK playing a secondary role and possibly participating in an as yet unidentified cellular process. It is remarkable that loss of wildtype AroK activity confers resistance to mecillinam [a bulky -lactam antibiotic that binds to E. coli penicillin-binding protein 2 (PBP2)], which is separable from the shikimate kinase activity of the enzyme. Although mecillinam sensitivity appears to be associated with AroK and not AroL, it may not be the primary function of the AroK protein. A number of other bacteria, for example, Haemophilus influenzae and Mycobacterium tuberculosis, encode only AroK but not AroL homologs. X-ray structure determination of AroK revealed a compact / protein (molecular dimensions 39 35 46 Å) that is structurally similar to Erwinia chrysanthemi SKII. The linear arrangement of the secondary structural elements within the polypeptide chain is 1, A, 2, B, C, D, 3, 310, E, 4, F, G, 5, and H (Fig. 1). The central part of the molecule, composed of a fivestranded parallel -sheet ( 5– 4– 1– 3– 2), is sandwiched between five -helices [Fig. 2(a)]. A cocrystal structure of E. chrysanthemi SKII and shikimate did not reveal a recognizable electron density feature corresponding to shikimate, but made it possible to implicate several charged residues in shikimate binding. All residues identified in this manner are structurally equivalent to those in AroK (Asp34, Arg58, Glu61, Gly78– 80, and Arg139 in E. chrysanthemi SKII correspond to Asp36, Arg60, Glu63, Gly81–83, and Arg140 in E. coli AroK). The putative shikimate-binding pocket is lined with additional hydrophobic side-chains that are likely to stabilize the binding of shikimate. One of these substratebinding site residues (Leu83 in E. chrysanthemi) is replaced with Lys86 in E. coli AroK [Fig. 3(a)]. We propose that loss of a stabilizing hydrophobic residue at this position may explain the significantly lower affinity of E. coli AroK for shikimic acid as compared to that of SKII. E. coli AroK contains motifs found in E. chrysanthemi SKII and many nucleotide-binding proteins (Fig. 1): Walker A(11–18: Gly–Pro–Met–Gly–Ala–Gly–Lys–Ser; consensus Gly–XXXX–Gly–Lys–Thr/Ser, where X is any residue) and B-motifs [77–82: Val–Leu–Ala–Thr–Gly–Gly; consensus ZZ–Asp–XX–Gly, where Z is a hydrophobic residue; in E. coli AroK the Asp residue is replaced with Ala; the function of the consensus Asp residue to coordinate Mg is taken over by Asp34 and Asp36; see Fig. 2(b)].. The adenine-binding motif found in the C-terminal portion of AroK differs significantly from the consensus sequence (Val/Ile–Asp–Ala–X–Gln/Asn–X–Pro) found in bacterial SKIIs and adenylyl kinase type-II isoenzymes. Although in AroK the corresponding polypeptide chain segment (152–159: Ile–Arg–Thr–Asp–Asp–Gln–Ser–Ala), which was not visible in our electron density maps, lacks several consensus residues, it is possible that this loop participates in adenine binding because nucleotide recognition depends solely on backbone interactions. Finally, the