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On the emergence of P-Loop NTPase and Rossmann enzymes from a Beta-Alpha-Beta ancestral fragment.

ABSTRACT: This article is dedicated to the memory of Michael G. Rossmann. Dating back to the last universal common ancestor, P-loop NTPases and Rossmanns comprise the most ubiquitous and diverse enzyme lineages. Despite similarities in their overall architecture and phosphate binding motif, a lack of sequence identity and some fundamental structural differences currently designates them as independent emergences. We systematically searched for structure and sequence elements shared by both lineages. We detected homologous segments that span the first ??? motif of both lineages, including the phosphate binding loop and a conserved aspartate at the tip of ?2. The latter ligates the catalytic metal in P-loop NTPases, while in Rossmanns it binds the nucleotide's ribose moiety. Tubulin, a Rossmann GTPase, demonstrates the potential of the ?2-Asp to take either one of these two roles. While convergence cannot be completely ruled out, we show that both lineages likely emerged from a common ??? segment that comprises the core of these enzyme families to this very day.

SUBMITTER: Longo LM

PROVIDER: S-EPMC7758060 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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On the emergence of P-Loop NTPase and Rossmann enzymes from a Beta-Alpha-Beta ancestral fragment.

Longo Liam M LM Jabłońska Jagoda J Vyas Pratik P Kanade Manil M Kolodny Rachel R Ben-Tal Nir N Tawfik Dan S DS

eLife 20201209

This article is dedicated to the memory of Michael G. Rossmann. Dating back to the last universal common ancestor, P-loop NTPases and Rossmanns comprise the most ubiquitous and diverse enzyme lineages. Despite similarities in their overall architecture and phosphate binding motif, a lack of sequence identity and some fundamental structural differences currently designates them as independent emergences. We systematically searched for structure and sequence elements shared by both lineages. We de ...[more]

PMID: 33295875

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Project description:Thermostability is the key to maintain the structural integrity and catalytic activity of enzymes in industrial biotechnological processes, such as terpene cyclase-mediated generation of medicines, chiral synthons, and fine chemicals. However, affording a large increase in the thermostability of enzymes through site-directed protein engineering techniques can constitute a challenge. In this paper, we used ancestral sequence reconstruction to create a hyperstable variant of the ent-copalyl diphosphate synthase PtmT2, a terpene cyclase involved in the assembly of antibiotics. Molecular dynamics simulations on the μs timescale were performed to shed light on possible molecular mechanisms contributing to activity at an elevated temperature and the large 40 °C increase in melting temperature observed for an ancestral variant of PtmT2. In silico analysis revealed key differences in the flexibility of a loop capping the active site, between extant and ancestral proteins. For the modern enzyme, the loop collapses into the active site at elevated temperatures, thus preventing biocatalysis, whereas the loop remains in a productive conformation both at ambient and high temperatures in the ancestral variant. Restoring a Pro loop residue introduced in the ancestral variant to the corresponding Gly observed in the extant protein led to reduced catalytic activity at high temperatures, with only moderate effects on the melting temperature, supporting the importance of the flexibility of the capping loop in thermoadaptation. Conversely, the inverse Gly to Pro loop mutation in the modern enzyme resulted in a 3-fold increase in the catalytic rate. Despite an overall decrease in maximal activity of ancestor compared to wild type, its increased thermostability provides a robust backbone amenable for further enzyme engineering. Our work cements the importance of loops in enzyme catalysis and provides a molecular mechanism contributing to thermoadaptation in an ancestral enzyme.

Dataset Information

On the emergence of P-Loop NTPase and Rossmann enzymes from a Beta-Alpha-Beta ancestral fragment.

Publications

On the emergence of P-Loop NTPase and Rossmann enzymes from a Beta-Alpha-Beta ancestral fragment.

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