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

Bryan J Jones,Nathan J Mylrea,Wendy R Gordon,Colin T Pierce,Debayan Chaudhury,Robert L Evans,Christine Luo,Romas J Kazlauskas,Bo Guan,Carrie M Wilmot,Oscar Millet

doi:10.1371/journal.pone.0235341

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 two-fold 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 50-fold, 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.

Highlights

Divergent evolution creates superfamilies of enzymes, which share the same protein fold, but differ in substrate specificity or in the type of catalytic activities
HNL1 and HbHNL share 81% identical amino acid residues over a comparison of 257 residues
There is some evidence for differences in active site conformations since the x-ray structure suggests increased flexibility of the residues in the substrate-binding site of HNL1 as compared to HbHNL

Summary

Introduction

Divergent evolution creates superfamilies of enzymes, which share the same protein fold, but differ in substrate specificity or in the type of catalytic activities. The focus of this paper is understanding how evolution creates new catalytic activity during divergent evolution. Duplication of the genes for these promiscuous enzymes followed by optimization of the promiscuous catalytic activity driven by increased organismal fitness is believed to give rise to enzymes with new primary activities. Support for this notion includes the observation that differing catalytic activities within a superfamily share mechanistic features or transition states and that enzymes within a superfamily often show promiscuous activities that correspond to the primary activities of other enzymes in the superfamily [4, 5]. Reconstructed ancestral enzymes have shown substrate promiscuity [6] and catalytic promiscuity [7, 8]

Methods

Results

Conclusion