Specificity

The model of the enzyme–triglyceride complex suggests that the enzyme may have a preference for binding the primary acyl chains in a position in which they can be hydrolysed. This is because access to the catalytic machinery would be difficult for a secondary chain due to steric constraints caused by the glyceride backbone.

The main difference in binding the two alternative primary positions of an acylglyceride is the direction in which the sn-2 chain points relative to the second glyceride carbon. As shown in Figures 3.8 and 3.9, the active site of AA7 appears to have hydrophobic patches on both sides of the substrate-binding cleft, mainly contributed by Trp 20 (at the top in the diagram) and Phe 250 (at the bottom), which could stabilise the sn-2 chain regardless of which primary chain is bound in the active site. This hydrophobic patch also appears to be able to stabilise the sn-2 chain of a glyceride bound in the opposite conformation, with the scissile chain in the P2 pocket and the backbone groups extending upwards out of the binding cleft, meaning that there are no obvious molecular features that could cause stereospecificity in AA7. It should be noted that it is not uncommon for lipases and esterases to be capable of hydrolysis at both of the primary positions of a glyceride; for example, human pancreatic lipase is active on both sn-1 and sn-3 positions of lipids (Jensen, deJong et al. 1983). The binding site of human pancreatic lipase is essentially symmetrical about the catalytic site (Egloff, Marguet et al. 1995), and, whereas the AA7 binding site is asymmetric, this is only because of the presence of the acceptor-binding P1 tunnel.

Selectivity in AA7 might be limited to regio- and typoselectivity dictating the direction in which a glyceride can bind. With the entire binding-site region being relatively hydrophobic, it would appear that either of the primary chains could be accommodated as long as they are less than the 10 carbon maximum size permitted by the P2 cleft. Further experiments with chiral inhibitors are under way to confirm if any stereoselectivity exists, and to explore the effects of chain size on binding direction and rates.

3.6.4.1 Determining Stereospecificity

Although not within the scope of the current work, several methods exist for

investigating the stereospecificity of the enzyme, and how different-sized acyl groups at different positions are able to bias this.

The first method involves analysis of the activity of the enzyme on different glyceride substrates, with different-sized substituents on each position of the glyceride. From this experiment, the liberation of the different-sized glycerides can be linked to the position from which they came. This would require some caution in drawing conclusions, however, as the stereospecificity of some enzymes can be biased

depending on the size of acyl groups at other positions. For example, certain bacterial lipases exhibit changes in sn-1 and sn-3 stereospecificity when the constituents present on the sn-2 position are varied (Stadler, Kovac et al. 1995).

An alternative involves using chirally pure glyceride-based inhibitors, such as that used in this study. By synthesising inhibitors with the phosphonate group at the three different chain positions, each inhibitor can be assayed for the rate at which it inhibits the enzyme. The inhibitor that matches the stereopreference of the enzyme will inhibit most efficiently. This work is currently being undertaken in a separate project (D. Colbert, personal communication, 2007).

3.7 Summary

The crystal structure of AA7 esterase from Lactobacillus rhamnosus was solved using

MAD methods to gain a low resolution (2.5 Å) structure, with a molecular

replacement solution on a native crystal providing a structure to a resolution of 1.7 Å.

This showed that, as anticipated from sequence analysis, AA7 is a single-domain αβ

hydrolase. In addition to the basic αβ hydrolase fold, the enzyme contains six inserted

helices, which are largely distributed around the substrate-binding regions of the enzyme. The enzyme contains a recognisable catalytic triad, composed of Ser–His– Asp, as is typical for these enzymes, with two distinct binding pockets that are bisected by the catalytic machinery. The structure of the enzyme in complex with a phosphonate-based tributyrin analogue suicide inhibitor was also solved by molecular replacement, showing that, with an inhibitor bound, the enzyme undergoes some conformational changes. These are primarily localised to helix D`, which, although bent into the substrate-binding site at the N-terminus in the native structure, becomes straightened upon binding of the substrate analogue in the inhibited structure, which removes the sidechain of Met 204 from its native position where it blocks most of the P2 binding pocket. With the catalytic machinery bisecting the binding cleft

perpendicularly, the catalytic histidine appears to be positioned to activate the

catalytic serine, as well as to activate a hydrolytic water that could enter the active site from either the P1 side or the P2 side. This bidirectional substrate binding is known to occur in Est2 from A. acidocaldarius, with the direction dependent on the substrate size, and recent data obtained in our group also appear to support this bidirectionality for AA7. The tunnel adjacent to the P1 binding site is postulated to be a binding site for an acceptor for the alcoholysis reaction. If this is the case, then alcoholysis could occur only when the substrate is oriented in such a way that the leaving group is in the P1 pocket, which, upon departure, would allow the acceptor to move forward from its binding tunnel to complete the deacylation of the enzyme and yield an ester product. This hypothesis, along with bidirectional substrate binding dependent on

typoselectivity, appears to provide an explanation for the differences in hydrolytic and alcoholytic rates of the enzyme on substrates of different sizes. More experiments to prove this hypothesis are currently being undertaken in our group.

Chapter 4

The Structure and

Mechanism of AZ4

Chapter 4

The Structure and Mechanism of AZ4 Esterase

4.1 Introduction

AZ4 is a carboxylesterase from Lactobacillus rhamnosus, identified primarily by its sequence similarity to the αβ carboxylesterase Est30 from Geobacillus

stearothermophilus (30% similarity) (Ewis, Abdelal et al. 2004). Prior to this work, AZ4 was cloned into a plasmid (pProEXHT-B, Invitrogen), with initial tests showing that it had hydrolytic activity on tributyrin agar, but not ester synthetic activity using tributyrin and an ethanol acceptor (M.-L. Delabre, personal communication, 2006). As the original scope of this project was to investigate the structural features of dairy lactic acid bacteria (LAB) that lead to substrate size preference, as well as

hydrolysis/alcoholysis preference, AZ4 was chosen for study as an example of a hydrolase-only enzyme.