Resurrected ancestral hydroxynitrile lyase from flowering plants
Comment
Authors show that resurrected ancestral plant esterases are as catalytically specific as modern esterases, that the ancestor of modern acetone cyanohydrin lyases was itself only very weakly promiscuous, and that improvements in lyase activity came at the expense of esterase activity
(Below N is a link to NCBI taxonomic web page and E link to ESTHER at designed phylum.) > other sequences: NE > artificial sequences: NE > synthetic construct: NE
Haloalkane_dehalogenase-HLD2 : 9zzzz-AncLinB Ancestral Haloalkane Dehalogenase AncLinB-DmbA, 9zzzz-AncFT7 Synthetic construct of Renilla-type engineered ancestral luciferase variant (AncFT7), 9zzzz-AncHLDRLuc2 Synthetic construct of the common ancestor of haloalkane dehalogenases and Renilla luciferase with fragment transplabntation (Anc-FT) AncFT, 9zzzz-AncHLDRLuc Synthetic construct of the common ancestor of haloalkane dehalogenases and Renilla luciferase
Molecular evidence
Database
No mutation 1 structure: 5TDX: Resurrected ancestral hydroxynitrile lyase from flowering plants No kinetic
LegendThis sequence has been compared to family alignement (MSA) red => minority aminoacid blue => majority aminoacid color intensity => conservation rate title => sequence position(MSA position)aminoacid rate Catalytic site Catalytic site in the MSA MATAHFVLIHTICHGAWIWYKLKPLLEAAGHKVTALDLAASGIDPRQIEQ INTFDEYSEPLLTFMESLPQGEKVILVGESCGGLNIALAADKYPEKISAA VFHNALMPDTEHSPSYVVDKFMEVFPDWKDTEFSTYTSNNETITGMKLGF KLMRENLYTNCPIEDYELAKMLTRKGSFFQNDLAQRPKFTEEGYGSIKRV YVWTDEDKIFPPEFQLWQIENYKPDKVYRVQGGDHKLQLSKTNELAEILQ EVADTYADLLAVAGLE
Hydroxynitrile lyases (HNL's) belonging to the alpha/beta-hydrolase-fold superfamily evolved from esterases approximately 100 million years ago. Reconstruction of an ancestral hydroxynitrile lyase in the alpha/beta-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 degrees 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-A resolution shows the expected alpha/beta-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.
The means by which superfamilies of specialized enzymes arise by gene duplication and functional divergence are poorly understood. The escape from adaptive conflict hypothesis, which posits multiple copies of a gene encoding a primitive inefficient and highly promiscuous generalist ancestor, receives support from experiments showing that resurrected ancestral enzymes are indeed more substrate-promiscuous than their modern descendants. Here, we provide evidence in support of an alternative model, the innovation-amplification-divergence hypothesis, which posits a single-copied ancestor as efficient and specific as any modern enzyme. We argue that the catalytic mechanisms of plant esterases and descendent acetone cyanohydrin lyases are incompatible with each other (e.g., the reactive substrate carbonyl must bind in opposite orientations in the active site). We then show that resurrected ancestral plant esterases are as catalytically specific as modern esterases, that the ancestor of modern acetone cyanohydrin lyases was itself only very weakly promiscuous, and that improvements in lyase activity came at the expense of esterase activity. These observations support the innovation-amplification-divergence hypothesis, in which an ancestor gains a weak promiscuous activity that is improved by selection at the expense of the ancestral activity, and not the escape from adaptive conflict in which an inefficient generalist ancestral enzyme steadily loses promiscuity throughout the transition to a highly active specialized modern enzyme.
Resurrected ancestral hydroxynitrile lyase from flowering plants
