Browsing by Author "Bennett, Matthew David"

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    The ester hydrolytic and synthetic activities of X-prolyl dipeptidyl peptidase from Streptococcus thermophilus : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science in Biochemistry at Massey University, New Zealand
    (Massey University, 2003) Bennett, Matthew David
    X-prolyl dipeptidyl peptidase (EC 3.4.14.11), or PepX, is a dipeptidase found in most dairy lactic acid bacteria that hydrolyses N-terminal dipeptides from larger peptides where proline is the residue penultimate to the scissile bond. It has recently been found that PepX will also catalyse the hydrolysis of some chromogenic esters and synthesise esters via an acyltransferase mechanism that uses ethanol as the acceptor molecule and tributyrin as the donor molecule. In this study, the pepX gene from Streptococcus thermophilus strain B2513 was cloned and sequenced. This sequence was found to differ in several positions from the recently published pepX sequence of S.thermophilus strain ACA-DC4. None of the observed substitutions occurred in the catalytic domain of the enzyme, all being localised to the C-terminal β-sheet domain. An activity assay using a chromogenic peptide substrate with tributyrin as an was used to prove that PepX binds peptide substrates and acylglycerides at the same binding site, implying that the same catalytic machinery carries out both peptide hydrolysis and activities involving acylglycerides. PepX was found to form esters only from the acylglyceride tributyrin, and was not active on any of the larger triglycerides tested. The chemical mechanism for this ester formation is proposed to involve the direct transfer of an acyl group from the donor to an acceptor, rather than acyl hydrolysis followed by the separate transfer of a carboxylic acid product onto an acceptor, as the enzyme does not form esters when provided with butyric acid and ethanol. PepX was found to be incapable of hydrolysing milkfat and tributyrin in aqueous solution. This contrasts with the ability of PepX to hydrolyse the synthetic ester p-nitrophenyl butyrate, which probably is a reflection of the lability of the ester bond in this substrate. The results of this study show that PepX is a peptidase that has a secondary acyltransferase activity, with no hydrolase activity on natural acylglyceride substrates.
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    The structure and function of esterases from lactic acid bacteria : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosphy in the Institute of Molecular BioSciences, Massey University, New Zealand
    (Massey University, 2007) Bennett, Matthew David
    Compounds derived from the breakdown of glyceride esters of milk fat, such as free fatty acids and short chain esters, are recognised as playing an important role in the flavour of a range of fermented foods. Esterases, capable of hydrolysing ester bonds, and in some cases, synthesising them via an acyltransferase mechanism, typically enter the fermentation from the starter and adjunct lactic acid bacteria that are used to inoculate milk to initiate the fermentation process. With such an important role in the development of both desirable and undesirable flavours, understanding how these enzymes operate is essential for product control. In this study, the crystal structures of three lactic acid bacterial esterases were solved: EstA from Lactococcus lactis, and AA7 from Lactobacillus rhamnosus which are both capable of hydrolysis of short chain triglycerides as well as synthesising esters via a transferase mechanism, and AZ4, an esterase from L. rhamnosus which appears to be limited to hydrolysis reactions. Whilst all three were found to be members of the hydrolase family, unique features were found for each enzyme, reflecting the large differences in their primary sequences, substrate specificities and activities. EstA and AA7 were both found to have a shallow substrate binding cleft, bisected by the catalytic machinery. The divided binding cleft suggests that during a transferase reaction the transferred group binds in one pocket, with the donor and acceptor groups (dependant on the stage of catalysis) binding in the other. In contrast, AZ4 was found to have a single deep substrate binding cavity, extending into the enzyme interior, with the catalytic residues located near its entrance. The absence of a second binding site for an acceptor is consistent with AZ4 having only one function – that of a hydrolase. The structures presented in this study are the first three dimensional structures of esterases from lactic acid bacteria to be reported. Their analyses, both in native form, and complexed with a varity of ligands mimicking various stages of the reaction cycle have highlighted how this basic fold can be adapted to efficiently catalyse different reactions. More importantly, in the case of AZ4, these structures have suggested that there is a novel mechanism used by the esterases to promote the enzyme reaction to proceed to completion, by preventing a futile catalytic reaction.