Exploiting the high-resolution crystal structure of Staphylococcus aureus MenH to gain insight into enzyme activity

The crystal structure of Staphylococcus aureus MenH has been determined at 2 ？ resolution. In the absence of a complex to inform on aspects of specificity a model of the enzyme-substrate complex has been used in conjunction with previously published kinetic analyses, site-directed mutagenesis studies and comparisons with orthologues to investigate the structure and reactivity of MenH.The overall basic active site displays pronounced hydrophobic character on one side and these properties complement those of the substrate. A complex network of hydrogen bonds involving well-ordered water molecules serves to position key residues participating in the recognition of substrate and subsequent catalysis. We propose a proton shuttle mechanism, reliant on a catalytic triad consisting of Ser89, Asp216 and His243. The reaction is initiated by proton abstraction from the substrate by an activated Ser89. The propensity to form a conjugated system provides the driving force for pyruvate elimination. During the elimination, a methylene group is converted to a methyl and we judge it likely that His243 provides a proton, previously acquired from Ser89 for that reduction. A conformational change of the protonated His243 may be encouraged by the presence of an anionic intermediate in the active site.Menaquinone, vitamin K2, is a lipid soluble quinone used by bacteria for electron transport. It is the sole quinone available to Gram-positive bacteria [1] whereas Gram-negative bacteria use ubiquinone under aerobic conditions, and menaquinone under anaerobic conditions [2]. Vitamin K, as menaquinone or the plant form phylloquinone, is a cofactor for γ-carboxylation of glutamate, a post-translational modification of proteins involved in blood coagulation and vascular biology and is an essential component of the mammalian diet [3].There are two distinct biosynthetic routes to menaquinone. One, recently discovered, occurs in a small subset of bacteria such as Helicobacter pylori [4]. The star