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

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 ?/? 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 ?/? hydrolase fold with its core ?/? 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.

SUBMITTER: Johnston JM

PROVIDER: S-EPMC3630204 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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

Johnston Jodie M JM Jiang Ming M Guo Zhihong Z Baker Edward N EN

PloS one 20130418 4

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 ...[more]

PMID: 23637813

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Project description:The PhoR/PhoB two-component system is a key regulatory protein network enabling Escherichia coli to respond to inorganic phosphate (Pi) starvation conditions by turning on Pho regulon genes for more efficient Pi uptake and use of alternative phosphorus sources. Under environmental Pi depletion, the response regulator (RR) component, PhoB, is phosphorylated at the receiver domain (RD), a process that requires Mg(2+) bound at the active site. Phosphorylation of the RD relieves the inhibition of the PhoB effector domain (ED), a DNA-binding region that binds to Pho regulon promoters to activate transcription. The molecular details of the activation are proposed to involve dimerization of the RD and a conformational change in the RD detected by the ED. The structure of the PhoB RD shows a symmetrical interaction involving alpha1, loop beta5alpha5 and N terminus of alpha5 elements, also seen in the complex of PhoB RD with Mg(2+), in which helix alpha4 highly increases its flexibility. PhoB RD in complex with Mg(2+) and BeF(3) (an emulator of the phosphate moiety) undergoes a dramatic conformational change on helix alpha4 and shows another interaction involving alpha4, beta5 and alpha5 segments. We have selected a series of constitutively active PhoB mutants (PhoB(CA)) that are able to turn on the Pho regulon promoters in the absence phosphorylation and, as they cannot be inactivated, should therefore mimic the active RD state of PhoB and its functional oligomerisation. We have analysed the PhoB(CA) RD crystal structures of two such mutants, Asp53Ala/Tyr102Cys and Asp10Ala/Asp53Glu. Interestingly, both mutants reproduce the homodimeric arrangement through the symmetric interface encountered in the unbound and magnesium-bound wild-type PhoB RD structures. Besides, the mutant RD structures show a modified active site organization as well as changes at helix alpha4 that correlate with repositioning of surrounding residues, like the active-site events indicator Trp54, putatively redifining the interaction with the ED in the full-length protein.

Dataset Information

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

Publications

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

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