Crystal structures of E. coli native MenH and two active site mutants

PLoS One. 2013 Apr 18;8(4):e61325. doi: 10.1371/journal.pone.0061325. Print 2013.

Abstract

Recent revision of the biosynthetic pathway for menaquinone has led to the discovery of a previously unrecognized enzyme 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase, also known as MenH. This enzyme has an α/β hydrolase fold with a catalytic triad comprising Ser86, His232, and Asp210. Mutational studies identified a number of conserved residues of importance to activity, and modeling further implicated the side chains of Tyr85 and Trp147 in formation of a non-standard oxyanion hole. We have solved the structure of E. coli MenH (EcMenH) at 2.75 Å resolution, together with the structures of the active site mutant proteins Tyr85Phe and Arg124Ala, both at 2.5 Å resolution. EcMenH has the predicted α/β hydrolase fold with its core α/β domain capped by a helical lid. The active site, a long groove beneath the cap, contains a number of conserved basic residues and is found to bind exogeneous anions, modeled as sulfate and chloride, in all three crystal structures. Docking studies with the MenH substrate and a transition state model indicate that the bound anions mark the binding sites for anionic groups on the substrate. The docking studies, and careful consideration of the active site geometry, further suggest that the oxyanion hole is of a conventional nature, involving peptide NH groups, rather than the proposed site involving Tyr85 and Trp147. This is in accord with conclusions from the structure of S. aureus MenH. Comparisons with the latter do, however, indicate differences in the periphery of the active site that could be of relevance to selective inhibition of MenH enzymes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Binding Sites
  • Catalytic Domain / genetics
  • Crystallography, X-Ray
  • Escherichia coli / enzymology
  • Models, Molecular
  • Molecular Docking Simulation
  • Oxo-Acid-Lyases / chemistry*
  • Oxo-Acid-Lyases / genetics
  • Oxo-Acid-Lyases / metabolism
  • Sequence Alignment

Substances

  • 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase
  • Oxo-Acid-Lyases

Grant support

This work was supported by the Ministry of Science and Innovation of New Zealand through the New Economy Research Fund (grant UOAX1005 to ENB), and by the Research Grants Council of the HKSAR government (grants GRF601209 and RPC11SC16 to ZG). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.