The 2.3-Å Crystal Structure of the Shikimate 5-Dehydrogenase Orthologue YdiB from Escherichia coli Suggests a Novel Catalytic Environment for an NAD-dependent Dehydrogenase

Abstract

We present here the 2.3-Å crystal structure of the Escherichia coli YdiB protein, an orthologue of shikimate 5-dehydrogenase. This enzyme catalyzes the reduction of 3-dehydroshikimate to shikimate as part of the shikimate pathway, which is absent in mammals but required for the de novo synthesis of aromatic amino acids, quinones, and folate in many other organisms. In this context, the shikimate pathway has been promoted as a target for the development of antimicrobial agents. The crystal structure of YdiB shows that the protomer contains two ␣/␤ domains connected by two ␣-helices, with the N-terminal domain being novel and the C-terminal domain being a Rossmann fold. The NAD ؉ cofactor, which co-purified with the enzyme, is bound to the Rossmann domain in an elongated fashion with the nicotinamide ring in the pro-R conformation. Its binding site contains several unusual features, including a cysteine residue in close apposition to the nicotinamide ring and a clamp over the ribose of the adenosine moiety formed by phenylalanine and lysine residues. The structure explains the specificity for NAD versus NADP in different members of the shikimate dehydrogenase family on the basis of variations in the amino acid identity of several other residues in the vicinity of this ribose group. A cavity lined by residues that are 100% conserved among all shikimate dehydrogenases is found between the two domains of YdiB, in close proximity to the hydride acceptor site on the nicotinamide ring. Shikimate was modeled into this site in a geometry such that all of its heteroatoms form high quality hydrogen bonds with these invariant residues. Their strong conservation in all orthologues supports the possibility of developing broad spectrum inhibitors of this enzyme. The nature and disposition of the active site residues suggest a novel reaction mechanism in which an aspartate acts as the general acid/base catalyst during the hydride transfer reaction.

Cloning, expression, purification and preliminary crystallographic characterization of a shikimate dehydrogenase fromCorynebacterium glutamicum

“…A presumably bifunctional SDH has also been found in Escherichia coli (YdiB; EC 1.1.1.282). This enzyme also reduces quinate to 3-dehydroquinate with either NADP or NAD as a cosubstrate (Benach et al, 2003). NAD is mostly found in catabolic reactions.…”

Site-directed Mutagenesis of the Active Site Region in the Quinate/Shikimate 5-Dehydrogenase YdiB of Escherichia coli

“…This portion of the protein possesses a unique ␣-␤-␣ sandwich motif, which also includes an ␣-helical hairpin structure at the C terminus of the protein and is further referred to as domain 1. The intervening sequence, domain 2, forms a Rossmann fold, to which the dinucleotide cofactor is bound with the A side of the nicotinamide ring facing the interdomain cleft (12,21,23). A direct comparison of the cofactor complexes for the two E. coli enzymes AroE and YdiB by Michel et al (12) revealed structural differences in their nucleotide-binding motifs, which likely account for the 10-fold higher affinity of YdiB for NAD than for NADP.…”

“…1 All of these structures reveal a common fold comprising two domains separated by a cleft. Although AroE from E. coli and AroE and YdiB from H. influenzae exist as monomers (12,22), YdiB from E. coli (12,23) and AroE from M. jannaschii (21) both homodimerize via their N-terminal domains. This portion of the protein possesses a unique ␣-␤-␣ sandwich motif, which also includes an ␣-helical hairpin structure at the C terminus of the protein and is further referred to as domain 1.…”

“…Recently, the crystal structures of AroE from E. coli, Meth-anococcus jannaschii, and H. influenzae (12,21,22) and of YdiB from E. coli (12,23) were solved in complex with NADP and NAD, respectively. Structures of apoenzymes are also available for H. influenzae AroE (22) and YdiB.…”

Automated streak-seeding with micromachined silicon tools

“…Expression and purification of Haemophilus influenzae hypothetical protein HI1161 and Xanthomonas campestris hypothetical protein XCC2852 (NESG targets IR63 and XcR50, 4 respectively) was carried out as part of the established high-throughput protein production pipeline of the NESG using previously published methods (Acton et al, 2005;Benach et al, 2003 ⎯ crystal structure of shikimate dehydrogenase).…”