Crystal Structure of a Novel Shikimate Dehydrogenase from Haemophilus influenzae

Abstract

To date two classes of shikimate dehydrogenases have been identified and characterized, YdiB and AroE. YdiB is a bifunctional enzyme that catalyzes the reversible reductions of dehydroquinate to quinate and dehydroshikimate to shikimate in the presence of either NADH or NADPH. In contrast, AroE catalyzes the reversible reduction of dehydroshikimate to shikimate in the presence of NADPH. Here we report the crystal structure and biochemical characterization of HI0607, a novel class of shikimate dehydrogenase annotated as shikimate dehydrogenase-like. The kinetic properties of HI0607 are remarkably different from those of AroE and YdiB. In comparison with YdiB, HI0607 catalyzes the oxidation of shikimate but not quinate. The turnover rate for the oxidation of shikimate is ϳ1000-fold lower compared with that of AroE. Phylogenetic analysis reveals three independent clusters representing three classes of shikimate dehydrogenases, namely AroE, YdiB, and this newly characterized shikimate dehydrogenase-like protein. In addition, mutagenesis studies of two invariant residues, Asp-103 and Lys-67, indicate that they are important catalytic groups that may function as a catalytic pair in the shikimate dehydrogenase reaction. This is the first study that describes the crystal structure as well as mutagenesis and mechanistic analysis of this new class of shikimate dehydrogenase.The shikimate pathway occupies a central position for aromatic biosynthesis in microbes and plants but is not present in humans and other higher animals. The absence of the shikimate pathway in animals makes it an ideal target for herbicide and anti-microbial drug design. Recently the shikimate pathway was identified in apicomplexan parasites, including Toxoplasma gondii and Plasmodium falciparum, which has renewed interest in better understanding the enzymes in the pathway (1, 2). The importance of the shikimate pathway is exemplified by the common herbicide glyphosate, which inhibits the enzyme 5-enolpyruvylshikimate-3-phosphate synthase. Recent studies have also shown that glyphosate blocks the shikimate pathway in apicomplexan parasites and is effective in controlling their growth (3). The shikimate pathway consists of seven enzymatic steps initiated by the condensation of phosphoenolpyruvate and erythrose-4-phosphate by 3-deoxy-Darabino-heptolosonate 7-phosphate synthase. The last enzymatic step produces the branch point intermediate, chorismate, which serves in turn as the precursor for a number of pathways including those involved in aromatic amino acid, phytoalexin, flavanoid, and lignin biosynthesis.The fourth enzyme in the pathway, shikimate dehydrogenase (shikimate:NADP ϩ oxidoreductase; EC 1.1.1.25), is involved in the NADPH-dependent reduction of dehydroshikimate to shikimate. This enzymatic reaction proceeds in both the forward and reverse direction with similar rates and similar Michaelis constants for substrates in either direction. Kinetic studies with substrate analogues and isotope exchange demonstrated that the shikimate dehydrogena...

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

“…It is highly specific for shikimate and for the cosubstrate NADP. A shikimate dehydrogenase-like enzyme (SDH-L; EC 1.1.1.25) from Haemophilus influenzae has been described that has comparable substrate and cosubstrate specificities to those of AroE, but that has a catalytic efficiency that is several orders of magnitude lower (Singh et al, 2005). A presumably bifunctional SDH has also been found in Escherichia coli (YdiB; EC 1.1.1.282).…”

“…Quinate is an abundant plant product and can be degraded by many bacteria and fungi (Grund & Kutzner, 1998). The structures of these different SDH subclasses are highly conserved (Singh et al, 2005), even if their sequence identitities are often only between approximately 25 and 35%.…”

“…In bacteria, SDH exists as a monomer or a homodimer (Singh et al, 2005;Chaudhuri & Coggins, 1985), while in plants SDH is associated with dehydroquinase as a bifunctional enzyme and in fungi it is part of the pentafunctional multienzyme complex catalyzing steps two to six of the shikimate pathway (Lumbsden & Coggins, 1977).…”

Regulation of Expression of Genes Involved in Quinate and Shikimate Utilization in Corynebacterium glutamicum

“…The CgR_1216 and CgR_1677 proteins exhibit less than 30% amino acid identity to E. coli NADPdependent shikimate dehydrogenase AroE in the shikimate biosynthetic pathway (1) and to YdiB, with an unknown physiological role (24). CgR_1216 exhibits 49% amino acid sequence identity to a Haemophilus influenzae shikimate dehydrogenase-like protein which has much less activity than E. coli AroE with shikimate as a substrate and has no activity on quinate (36). CgR_1677 exhibits 49% amino acid identity to Mycobacterium tuberculosis NADP-dependent shikimate dehydrogenase (10).…”

“…Unrooted phylogenetic tree showing relationships among bacterial shikimate dehydrogenase family proteins and the homologues in C. glutamicum. The proteins analyzed were E. coli AroE and YdiB (AroE-Ec and YdiB-Ec) (1,24), H. influenzae AroE and SdhL (AroE-Hi and SdhL-Hi) (36,47), M. tuberculosis shikimate dehydrogenase (SDH-Mt) (10), Amycolatopsis mediterranei RifI (RifI-Am) (48), and C. glutamicum QsuD, CgR_1216, and CgR_1677, encoded by qsuD, cgR_1216, and cgR_1677, respectively. The amino acid sequences were aligned using the CLUSTAL W program (40), and the tree was constructed using the neighbor-joining method (31).…”