A fundamental catalytic difference between zinc and manganese dependent enzymes revealed in a bacterial isatin hydrolase

Theis Sommer,Jürgen Gauss,Michael Etzerodt,J Preben Morth,Gregor Diezemann,Lalita Uribe,Kaare Bjerregaard-Andersen,Michele Cascella

doi:10.1038/s41598-018-31259-y

Abstract

The catalytic mechanism of the cyclic amidohydrolase isatin hydrolase depends on a catalytically active manganese in the substrate-binding pocket. The Mn2+ ion is bound by a motif also present in other metal dependent hydrolases like the bacterial kynurenine formamidase. The crystal structures of the isatin hydrolases from Labrenzia aggregata and Ralstonia solanacearum combined with activity assays allow for the identification of key determinants specific for the reaction mechanism. Active site residues central to the hydrolytic mechanism include a novel catalytic triad Asp-His-His supported by structural comparison and hybrid quantum mechanics/classical mechanics simulations. A hydrolytic mechanism for a Mn2+ dependent amidohydrolases that disfavour Zn2+ as the primary catalytically active site metal proposed here is supported by these likely cases of convergent evolution. The work illustrates a fundamental difference in the substrate-binding mode between Mn2+ dependent isatin hydrolase like enzymes in comparison with the vast number of Zn2+ dependent enzymes.

Highlights

The catalytic activity of metallohydrolase enzymes strongly depends on the identity of the metal and on the nature of its binding site
The mechanism of a Mn2+ dependent hydrolysis reaction is characterised in detail by computer simulations using multiscale quantum mechanics/molecular mechanics (QM/MM)[22] and ab initio molecular dynamics (MD) simulations[23]
Based on the identification of mechanistically important residues presented in this work, including the Mn2+ binding residues in the LaIHA active site, we further present evidence that the Mn2+ dependent allantoate amidohydrolase (AAH) (E.C. 3.5.3.9) displays a striking similarity in the active site geometry despite the differences in the overall protein fold

Summary

Introduction

The catalytic activity of metallohydrolase enzymes strongly depends on the identity of the metal and on the nature of its binding site. Isatin hydrolase A from Labrenzia aggregata (LaIHA) and R. solanacearum (RsIHA) share 59% sequence identity while the two putative orthologues LaIHA and LaIHB in L. aggregata share 51% sequence identity Both LaIHA and LaIHB contain the central metal binding motif HxG[T/A]HxDxPxH in each protomer of the catalytically active dimer. Based on the identification of mechanistically important residues presented in this work, including the Mn2+ binding residues in the LaIHA active site, we further present evidence that the Mn2+ dependent allantoate amidohydrolase (AAH) (E.C. 3.5.3.9) displays a striking similarity in the active site geometry despite the differences in the overall protein fold As this likely represents a case of convergent evolution, we propose that the generalised catalytic mechanism described here for the IHs may apply to the AAH family and other similar Mn2+ dependent hydrolases

Methods

Results

Conclusion