Structural Comparison with Actinidia eriantha Carboxylesterase

Recently, the crystal structure of AeCXE1, an HSL class carboxylesterase from Actinidia eriantha (kiwifruit), was published (Ileperuma, Marshall et al. 2007). Despite no significant similarity in sequence, as expected from the large phylogenetic divergence of the source species, and the fact that the enzymes are members of different lipase classes, this structure has some remarkably similar features to that of AZ4. Both enzymes have a “cap” that forms the top of the substrate-binding site. In AZ4, this cap is made up of helix D` and extended helix D, whereas, in AeCXE1, the cap is made partially from inserted elements in the region of helix D and partially from an extended loop contributed from the N-terminus of the protein. Despite these differences in the formation of the cap, the end result is a very similar surface topology in this region, as shown in Figure 4.14. This suggests that the shape of the binding sites in αβ hydrolases might be subjected to conservation more than the

Figure 4.14 Paraoxon bound AZ4 (grey) and paraoxon bound AeCXE1 from A.

eriantha (yellow). Left panel: Cartoon comparison contrasting secondary

structural differences, seen mainly at the top of the molecule. Right panels: Surface map of individual structures, showing the similar surface topography. The bound paraxon residues are shown as sticks, visible in the catalytic-site cleft. The colouring is the same as for the main panel.

4.7 Summary

From the structures obtained, much information about the mechanism of catalysis and substrate binding of AZ4 has been ascertained. Like all other αβ hydrolases, AZ4 uses

a catalytic triad made of a nucleophile, a base and an acid, with the nucleophile located in an unfavourable Ramachandran conformation in a nucleophile elbow. The resting state structure of the enzyme was solved, and showed that the substrate- binding cavity and the cleft are preformed, rather than being induced by substrate association with the enzyme. Comparison of this structure with the structure of the high molecular weight lipase from C. rugosa (Grochulski, Bouthillier et al. 1994) showed that there were similarities in the architecture of the cleft, suggesting that AZ4 utilises a similar “tuning fork” substrate-binding conformation, with the scissile acyl chain projecting into the cavity and the other two chains of the triglyceride

accommodated in the cleft on the enzyme surface.

In the resting state, the serine nucleophile is in a conformation that places it too far from the catalytic His 219 to form a hydrogen bond with it, a prerequisite for

activation of the nucleophile. In the structure solved from crystals soaked in NaCl and tributyrin, the serine nucleophile was seen in a split conformation, one of which was

close enough and in an appropriate geometry to form a hydrogen bond with the Nε of

His 283.

Analysis of the structure obtained from crystals soaked in paraoxon provided information about the formation of a tetrahedral transition state and details of the possible mechanism. Most importantly, the change in conformation of the serine nucleophile shows that, either during the nucleophilic attack or after it, the serine sidechain rotates back to a conformation close to that seen in the resting state. This movement takes the serine out of hydrogen-bonding range of the histidine and appears to ensure that the reaction proceeds in a productive direction, by preventing the passage of the proton from His 219 back to Ser 94.

219 and to the mainchain amide of the catalytic serine. Such product binding has not been previously observed in αβ hydrolases. It is postulated that it is this interaction

that induces the serine to change conformation from its usual unfavourable mainchain configuration into a helical conformation. As well as inducing the change in the serine, the formation of the hydrogen bond between the serine amide and the carboxylate oxygen breaks the bond that was previously present between the serine amide and the carbonyl oxygen of Phe 116. This causes a conformational change in the loop region between Phe 116 and His 126, which results in the rotation of Ile 120 into the binding cavity, effectively filling it completely. Although this movement is not sufficient to cause an acetate or butyrate product to become dissociated from the enzyme (under the conditions used for crystal soaking), it may have a role in assisting the ejection of larger molecules from the enzyme.

Chapter 5

Chapter 5

Concluding Remarks

5.1 Summary

This study has answered many of the long-standing questions about the structure of tributyrin esterases from dairy lactic acid bacteria, showing that, as anticipated from sequence analyses, they are all members of the αβ hydrolase family. Despite this

similarity, however, all three enzymes examined have unique features that will help to advance understanding of how each enzyme functions in an industrial context, as well as to increase knowledge of the αβ hydrolase family in general.

5.1.1 Structural Elucidation

The crystal structures of three different esterases, EstA from L. lactis, and AA7 and

Az4 from Lb. rhamnosus, were solved, and complexes of AA7 and AZ4 with suicide

inhibitors or ligands bound were also determined.

Overall, all three enzymes conform to the basic αβ hydrolase fold that is typical of

many esterases and lipases. Compared with the prototype fold, they all contain inserted helices in locations that are typical for deviations from the basic fold. No additional β strands are seen in the main sheet, a phenomenon that tends to be limited

to lipases.

The substrate-binding elements of all three enzymes are found either on the loops between the secondary structural elements of the basic fold or on the inserted

secondary elements, in line with the observation that the αβ hydrolase enzyme acts as

a framework that allows modification for specific catalytic roles (Ollis, Cheahet al. 1992).

Although the enzymes catalyse reactions on similar substrates, analyses of the

structures have shown that they are sufficiently different that a detailed comparison of all three structures is virtually impossible.

The solutions of the structures of these three enzymes nonetheless provide additional insights into the relationship between esterase structure and function. All three

proteins share a similar fold, yet each has a differing arrangement of substrate-binding pockets, showing that different approaches can be used to catalyse different reactions. All three structures also show unique features that add to the understanding of the αβ

hydrolase family in general.

One of the key differences in the activities of the three enzymes is that, whereas AA7 and EstA have been shown to be capable of hydrolysing ester bonds and also of synthesising esters through a transferase reaction, AZ4 is believed to be capable only of hydrolysis. This difference in activity is reflected in the arrangement of the substrate-binding sites of each enzyme.

5.2 Structural Summaries

5.2.1 AZ4

AZ4 was found to have an αβ hydrolase fold, although strand 1 of the prototype fold

is absent. It has several insertions to the basic fold, with the most significant being between strand 6 and helix D, consisting of a loop that forms part of the substrate- binding cavity and a large cap over the top of the active site. The cap is also involved in forming a crystallographic homodimer through a symmetry operation. The native crystals and the crystal-soaking experiments produced structures that appear to represent the enzyme in resting, activated, intermediate and product-bound states. From these structures, it was found that the nucleophilic serine sidechain undergoes

donating the proton back to the serine. A different mainchain conformation was observed in the product-bound state, where, because of rotation of the preceding peptide bond, the serine is in a helical conformation, which is uncharacteristic of αβ

hydrolases. This change appears to trigger a further conformational change in the loop that is inserted between strand 6 and helix D, which closes the substrate-binding site. It is easy to imagine that such a conformational change might have a role in assisting the dissociation of a substrate at the end of the catalytic cycle, providing a novel “self- ejection” mechanism. The conformational changes observed have not been seen in any other αβ hydrolase family members to date, although the closest relative of AZ4

with a structure solved, Est30 from Bacillus stearothermophilus (Liu, Wang et al. 2004), has a very similar inserted loop, which is likely to be capable of undergoing a similar change.

5.2.1.1 AZ4 Binding-site Topology

There appears to be no acceptor-binding site in AZ4. The enzyme has a single binding cleft, with the catalytic histidine exposed to bulk solvent, allowing easy access of water during hydrolysis (Figure 5.1). This likely reflects the fact that AZ4 has been shown to be strictly a hydrolytic enzyme, and therefore does not require a specific binding site for an acceptor molecule, with hydrolytic water able to directly enter the active site from solution.

Interestingly, the arrangement of the binding site of AZ4 superficially resembles that of Actinidia eriantha AeCXE1 carboxylesterase, despite the enzymes being

apparently unrelated (other than both being αβ hydrolases), demonstrating convergent

evolution of binding-site topology for a similar function.