Larger active site in an ancestral hydroxynitrile lyase increases catalytically promiscuous esterase activity

Abstract

Hydroxynitrile lyases (HNL's) belonging to the α/β-hydrolase-fold superfamily evolved from esterases approximately 100 million years ago. Reconstruction of an ancestral hydroxynitrile lyase in the α/β-hydrolase fold superfamily yielded a catalytically active hydroxynitrile lyase, HNL1. Several properties of HNL1 differ from the modern HNL from rubber tree (HbHNL). HNL1 favors larger substrates as compared to HbHNL, is twofold more catalytically promiscuous for ester hydrolysis (p-nitrophenyl acetate) as compared to mandelonitrile cleavage, and resists irreversible heat inactivation to 35˚C higher than for HbHNL. We hypothesized that the x-ray crystal structure of HNL1 may reveal the molecular basis for the differences in these properties. The x-ray crystal structure solved to 1.96-Å resolution shows the expected α/β-hydrolase fold, but a 60% larger active site as compared to HbHNL. This larger active site echoes its evolution from esterases since related esterase SABP2 from tobacco also has a 38% larger active site than HbHNL. The larger active site in HNL1 likely accounts for its ability to accept larger hydroxynitrile substrates. Site-directed mutagenesis of HbHNL to expand the active site increased its promiscuous esterase activity 50fold, consistent with the larger active site in HNL1 being the primary cause of its promiscuous esterase activity. Urea-induced unfolding of HNL1 indicates that it unfolds less completely than HbHNL (m-value = 0.63 for HNL1 vs 0.93 kcal/mol�M for HbHNL), which may account for the ability of HNL1 to better resist irreversible inactivation upon heating. The structure of HNL1 shows changes in hydrogen bond networks that may stabilize regions of the folded structure.

Biocatalysis making waves in organic chemistry

“…The synthesis then is a biocatalytic Henry reaction (Scheme 4). 40,41,47,48 Recent developments have focussed on even more enzymes from unusual sources such as millipedes 44,[51][52][53] and bacteria, [54][55][56] the exploration of evolutionary ancestors 57 and other methods to interconvert HNL activity with other enzyme activities, in particular hydrolase activity. 58 Reaction engineering with a special focus of the application of HNLs in flow has recently led to promising results.…”

Engineering of Ancestors as a Tool to Elucidate Structure, Mechanism, and Specificity of Extant Terpene Cyclase

“… 13 Crystal structures of reconstructed enzymes have been presented and been used together with biochemical analyses to derive knowledge about principles of molecular evolution. 11 , 19 − 25 We anticipated that a reconstructed thermostable ancestral SvS (SvS-A2) 26 would be more amenable to crystallization and could serve as structural template to derive a homology model of the extant enzyme. In this study, we show that further engineering of a surface patch in the reconstructed ancestor was necessary to achieve this goal.…”

“…Since ancestral sequence reconstruction grants access to functional and robust enzymes without requiring structural input, it constitutes a powerful engineering approach , alongside other existing methods to enhance stability and solubility. , As protein crystallization benefits from high stability and solubility, , it has been suggested that structures of ancestral enzymes could be used as a platform to approach the structures of extant enzymes that are challenging to study . Crystal structures of reconstructed enzymes have been presented and been used together with biochemical analyses to derive knowledge about principles of molecular evolution. ,− We anticipated that a reconstructed thermostable ancestral SvS (SvS-A2) would be more amenable to crystallization and could serve as structural template to derive a homology model of the extant enzyme. In this study, we show that further engineering of a surface patch in the reconstructed ancestor was necessary to achieve this goal.…”

Substrate Dynamics Contribute to Enzymatic Specificity in Human and Bacterial Methionine Adenosyltransferases

“… 15 While the role of protein structural dynamics in this process has been described, the role of substrate conformational sampling is comparatively poorly understood. 16 It has recently been reported that large active sites can accommodate multiple different productive substrate conformations without changing the conformation of the catalytic pocket 17 , 18 and that in some cases new Michaelis complexes can be recognized. 19 …”