Structure and function of the alpha beta hydrolase MhpC

• Main Author: Burakorn, Jiraporn
• Other Authors: Wood, S. P. ; Cooper, J. B.
• Published: University of Southampton 2009
• Subjects: 572.7; QH301 Biology
• Online Access: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.503205

2-hydroxy-6 keto nona 2, 4 diene 1,9 dioic acid 5,6 hydrolase (MhpC) is a homodimeric enzyme belonging to the α/β hydrolase fold family. The three catalytic residues comprise Ser110, Asp235 and His263. To study structure and catalytic mechanism of MhpC, Ser110 was replaced by cysteine and aspartic acid. A range of other mutants were also investigated. The S110C mutant enzyme showed 400 fold reduced catalytic efficiency compared with wild type enzyme, implying that it is a crucial residue in catalysis. The crystal structure of MhpC S110C demonstrated a water molecule interleaving between Cys110 and His263. Exposure of mutant enzyme crystals to substrate showed formation of a covalent complex with the product (hydroxypentadienoic acid) bound through C3 probably by a Michael adduct reaction with the Cys110 thiol. The enzyme-product adduct was confirmed by mass spectrometry. Interestingly, the intact enzyme-substrate complex was also observed by mass spectrometry. The S110D mutant enzyme was not active. The crystal structure of MhpC S110D showed that the active site residue His263 was slightly moved, forming a good hydrogen bond with D110, and many residues in the active site region (Trp264, Phe173 and Phe237) were rearranged. The carboxylate group may provide an interesting mimic of a gem-diol transition state. Serine 40 is close to the catalytic triad and S40C enzyme was 18 fold less active than wild type enzyme. The crystal structure of MhpC S40C revealed an interleaving water molecule between Ser110 and His263. The S40C/S110C double mutant succeeded to form a disulphide bridge redox-switch in the enzyme active site. To disrupt half-of-sites reactivity of MhpC a number of residues were mutated at the subunit interface of the dimeric enzyme. Radical changes in residue type led to mis-folding of MhpC. The more conservative N109G/H256N double mutant enzyme was correctly folded and dimeric although a charged substituent was no longer present in the hydrogen bonding network at the interface. Titration of charged interface residues showed monomeric MhpC by gel filtration at low pH but enzyme activity is low in these conditions.