Crystal structure of an inferred ancestral bacterial pyruvate decarboxylase

Abstract

An ancestral bacterial pyruvate decarboxylase (with an inferred age of 1248 million years) was reconstructed through ancestral sequence reconstruction, synthesized and recombinantly expressed in E. coli. The enzyme is fully functional and its crystal structure was elucidated to 3.5 Å resolution.

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

“…The fact that the ancestral enzyme yields a crystal structure is in line with the presumption that ancestral enzymes could be more amenable for structural characterization, 13 and in agreement with reports of other metabolic enzymes for which ancestral crystal structures have been reported, such as a bacterial pyruvate decarboxylase and mammalian cytochrome P450. 20 , 48 …”

“…The fact that the ancestral enzyme yields a crystal structure is in line with the presumption that ancestral enzymes could be more amenable for structural characterization, 13 and in agreement with reports of other metabolic enzymes for which ancestral crystal structures have been reported, such as a bacterial pyruvate decarboxylase and mammalian cytochrome P450. 20,48 Using Ancestral Enzyme Structure as Template for Homology Modeling of Extant SvS. Terpene cyclases that share a similar active site fold have been shown to exhibit low sequence identity.…”

“…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.…”

To beat the heat – engineering of the most thermostable pyruvate decarboxylase to date

“…Ancestral sequence reconstruction, supposedly a powerful method of resurrecting ancient thermostable enzymes, did not yield more thermostable PDC. 7 A computational Rosetta-based design resulted in several variants with improved stability, determined by differential interference contrast (DIC) microscopy; unfortunately, the work was not corroborated by kinetic activity and stability data. 8 Another effort to redirect the specicity of a thermostable, acetolactate synthase to perform a PDC-like reaction by conventional directed evolution did not yield variants either, with activity similar to that of bacterial PDC.…”