Journal of Molecular Biology
Volume 346, Issue 1, 11 February 2005, Pages 253-265
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The Structure of the C–C Bond Hydrolase MhpC Provides Insights into its Catalytic Mechanism

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2-Hydroxy-6-ketonona-2,4-diene-1,9-dioic acid 5,6-hydrolase (MhpC) is a 62 kDa homodimeric enzyme of the phenylpropionate degradation pathway of Escherichia coli. The 2.1 Å resolution X-ray structure of the native enzyme determined from orthorhombic crystals confirms that it is a member of the α/β hydrolase fold family, comprising eight β-strands interconnected by loops and helices. The 2.8 Å resolution structure of the enzyme co-crystallised with the non-hydrolysable substrate analogue 2,6-diketo-nona-1,9-dioic acid (DKNDA) confirms the location of the active site in a buried channel including Ser110, His263 and Asp235, postulated contributors to a serine protease-like catalytic triad in homologous enzymes. It appears that the ligand binds in two separate orientations. In the first, the C6 keto group of the inhibitor forms a hemi-ketal adduct with the Ser110 side-chain, the C9 carboxylate group interacts, via the intermediacy of a water molecule, with Arg188 at one end of the active site, while the C1 carboxylate group of the inhibitor comes close to His114 at the other end. In the second orientation, the C1 carboxylate group binds at the Arg188 end of the active site and the C9 carboxylate group at the His114 end. These arrangements implicated His114 or His263 as plausible contributors to catalysis of the initial enol/keto tautomerisation of the substrate but lack of conservation of His114 amongst related enzymes and mutagenesis results suggest that His263 is the residue involved. Variability in the quality of the electron density for the inhibitor amongst the eight molecules of the crystal asymmetric unit appears to correlate with alternative positions for the side-chain of His114. This might arise from half-site occupation of the dimeric enzyme and reflect the apparent dissociation of approximately 50% of the keto intermediate from the enzyme during the catalytic cycle.

Introduction

2-Hydroxy-6-ketonona-2,4-diene-1,9-dioic acid 5,6-hydrolase (MhpC) from Escherichia coli catalyses the cleavage of the C5–C6 bond of 2-hydroxy-6-keto-nona-2,4-diene-1,9-dioic acid (2), a dienol ring fission product (RFP) of the bacterial meta-cleavage pathway for degradation of phenylpropionic acid (Figure 1). Several enzymes catalysing rather similar reactions have been characterised as 2-hydroxy-6-oxo-dienoic acid (HODA) hydrolases producing a common product, 2-hydroxypenta-2,4-dienoic acid (5), and variable other products depending upon the size and character of the C6 substituent of the substrate. In the case of 2-hydroxy-6-keto-6-phenylhexa-2,4-dienoic acid hydrolase (BphD), which cleaves the RFP of biphenyl, the second product is benzoic acid,1 whereas 2-hydroxy-6-oxohepta-2,4-dienoate hydrolase (TodF) from the toluene degradation pathway produces acetic acid.2 For MhpC, there is a propionic acid substituent at C6 of the substrate and this generates succinic acid (6) as the second product. This cleavage of a C–C bond by MhpC and a selection of its distant sequence homologues is a relatively rare enzyme-catalysed reaction, shared by a wider group of enzymes termed the β-ketolases.3

The HODA hydrolases are members of the α/β hydrolase family of enzymes, a group sharing common features of three-dimensional structure but without extensive sequence homology.4, 5 The catalytic specificities of the family enzymes vary widely although rather similar topological arrangements of some active-site residues are observed. A conserved triad of presumptive nucleophile (serine or aspartate), acid and histidine residues occupy similar locations with respect to a common motif termed the nucleophile elbow, a feature well characterised in the serine hydrolases. There is experimental evidence that the conserved serine residue may function in some α/β hydrolases as a protein nucleophile as it does in serine proteases.6, 7 However, kinetic and 18O incorporation experiments suggest that for the MhpC hydrolase an enzyme-activated water molecule may form a gem-diol intermediate,8, 9, 10 reminiscent of the aspartic proteinases.11 A mechanism for the MhpC catalysed cleavage reaction proposed by Bugg involves an enol/keto tautomerisation to provide a keto intermediate (3) that acts as an electron sink for C–C bond cleavage via an enzyme-activated nucleophile, X (Figure 1). This nucleophile could be an activated water molecule or an amino acid side-chain.

To define the active site of the enzyme and initiate a resolution of this issue, we have determined the 3D structure of native MhpC hydrolase and analysed the structure of the complex formed by co-crystallising the enzyme with a non-hydrolysable substrate analogue, 2,6-diketo-nona-1,9-dioic acid (DKNDA), and with a product analogue, laevulinic acid. Although a number of α/β hydrolase structures have been determined, none proved effective as search models for molecular replacement and therefore we determined the structure using selenium incorporation and multi-wavelength anomalous dispersion methods.

Section snippets

Native structure solution

The expression system for MhpC was effective, producing approximately 100 mg of enzyme per litre of culture with or without selenomethionine incorporation. Gel-filtration chromatography and SDS-PAGE confirmed that the enzyme was a dimer of 31,000 Da subunits. Crystals of native MhpC were formed under a selection of different screen conditions but, in general, these did not produce crystals in the presence of substrate or product analogues. New conditions were required in each case. The

Active-site structure and catalytic mechanism

The overall protein fold for MhpC is very similar to those reported for HODA enzymes BphD,16 CarC20 and CumD.23 Sequence and structural homology at the active sites of α/β C–C hydrolases involving an active Asp-His-Ser triad has led to a widespread belief that a serine nucleophile is involved in the mechanism in much the same way as it is in serine proteases. Mutagenesis of the serine residue and subsequent loss of activity has supported this hypothesis.7, 25 However, detailed investigations of

Protein preparation and crystallisation

Native MhpC hydrolase was overexpressed in E. coli BL21(DE3)pLysS carrying the plasmid pIPC by IPTG-induction of log phase cultures as described.10 Typically, a 10 l fermentation produced 50 g of cells. These were lysed by sonication at 4 °C in 50 mM potassium phosphate buffer (pH 7.5) containing 5 mM 2-mercaptoethanol. Enzyme was precipitated from the clarified lysate by precipitation in ammonium sulphate (25% saturation at 4 °C) for four hours. The precipitated protein was resuspended in phosphate

Acknowledgements

This work was supported by the BBSRC and the Wellcome Trust.

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    Present addresses: F. Mohammed, Division of Cancer Studies, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK; T. Robertson, Department of Medicine, University of Queensland, Princess Alexandra Hospital, Woolloongabba, Qld, 4102, Australia.

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