Structural and evolutionary insights from the reconstructed ancestral plant hydroxynitrile lyase, HNL1

Abstract

HNL1 is a reconstructed flowering plant hydroxynitrile lyase that is believed to have existed about 100 million years ago and displays promiscuity for esterase functionality. In other words, although HNL1 has low activity for an esterase function that hydrolyzes esters to an alcohol and acids, it is primarily a hydroxynitrile lyase, catalyzing the breaking of acetone cyanohydrin to acetone and cyanide (independent of water). The cyanide, produced through HNL1's lyase function, is released when herbivores wound the plant, thus acting as a natural defense mechanism. HNL1 allows us to see divergence in the evolutionary history of the alpha/beta‐hydrolase fold superfamily, a general class of hydrolytic enzymes with a core of variable numbers of alpha helices and beta strands. Indeed, HNL1 has a core of six alpha helices and six beta strands, and because it gave rise to several modern hydroxynitrile lyases (in plants such as the rubber tree, cassava, and wild castor) while retaining methyl esterase activity, we can explore the divergence between these two types of enzymes on the structural level. Specifically, we investigated how the reconstructed ancestral protein HNL1 phylogenetically relates to modern HNL and esterase proteins. The Minnetonka CBM SMART team used 3D modeling and printing technology to examine structure‐function relationships and the divergence of HNL1. Through the use of the Basic Local Alignment Search Tool (BLAST) provided by the National Center for Biotechnology Information (NCBI), we determined the percent similarity between protein sequences of the ancestral protein HNL1 and its modern counterparts. Then, this information was used to construct a phylogenetic tree in which HNL1's position at a specific node of special divergence was identified. Finally, while it is known that some of the amino acids in the active site are important for promiscuity, the specific identity and location of these amino acids within the protein are still actively being researched.