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
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.