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

Karen Schriever,Natalie M Hendrikse,Elton P Hudson,Antonino Biundo,Robert Schnell,Per-Olof Syrén,Patricia Saenz-Mendez,Reshma Srilakshmi Rudraraju

doi:10.1021/jacs.0c10214

Abstract

Structural information is crucial for understanding catalytic mechanisms and to guide enzyme engineering efforts of biocatalysts, such as terpene cyclases. However, low sequence similarity can impede homology modeling, and inherent protein instability presents challenges for structural studies. We hypothesized that X-ray crystallography of engineered thermostable ancestral enzymes can enable access to reliable homology models of extant biocatalysts. We have applied this concept in concert with molecular modeling and enzymatic assays to understand the structure activity relationship of spiroviolene synthase, a class I terpene cyclase, aiming to engineer its specificity. Engineering a surface patch in the reconstructed ancestor afforded a template structure for generation of a high-confidence homology model of the extant enzyme. On the basis of structural considerations, we designed and crystallized ancestral variants with single residue exchanges that exhibited tailored substrate specificity and preserved thermostability. We show how the two single amino acid alterations identified in the ancestral scaffold can be transferred to the extant enzyme, conferring a specificity switch that impacts the extant enzyme’s specificity for formation of the diterpene spiroviolene over formation of sesquiterpenes hedycaryol and farnesol by up to 25-fold. This study emphasizes the value of ancestral sequence reconstruction combined with enzyme engineering as a versatile tool in chemical biology.

Highlights

Understanding how biosynthetic enzymes assemble chiral, complex products[1−3] from simpler metabolites requires structural information on active site architectures
We previously reported a hypothetical ancestor of Spiroviolene synthase (SvS) SvSA2 (Figure S1)[26] which shares 77% sequence identity with the extant enzyme
Half of all FDA-approved drugs are based on natural products or derivatives thereof, and understanding how nature assembles its array of chiral, complex structures from simpler metabolites is a long-standing goal in enzymology and synthetic biology.[2,3,65,66]

Summary

Introduction

Understanding how biosynthetic enzymes assemble chiral, complex products[1−3] from simpler metabolites requires structural information on active site architectures. A mechanism for spiroviolene formation has been suggested based on NMR experiments;[4] yet due to the lack of a crystal structure it has remained unresolved how the enzyme chaperones the linear substrate in its active site during the cyclization reaction leading to the spirocyclic terpene. We have obtained a crystal structure of a stable and soluble reconstructed ancestor of SvS and used an engineered crystallized variant thereof as a template to derive a highconfidence homology model of extant SvS. Structural information enabled us to understand the molecular basis of substrate promiscuity and to engineer substrate specific variants of both ancestral and extant SvS. Crystal structures of reconstructed enzymes have been presented and been used together with

Objectives

Results

Conclusion