Neutron/X‐ray Joint Refinement of The Active and Inactive Form of Helicobacter pylori 5′‐ Methylthioadenosine Nucleosidase

Abstract

5′‐Methylthioadenosine nucleosidase (HpMTAN) is an essential Helicobacter pylori enzyme that hydrolyzes N‐ribosidic bonds of a variety of adenosine‐based metabolites. Recently, HpMTAN has become an attractive drug target due to the enzyme's role in the menaquinone pathway that is only specific for H. pylori and Campylobacter. In the proposed catalytic mechanism of HpMTAN, the reaction is initiated by D198 that acts as a general acid to protonate the N7 position of the adenine moiety. The protonation creates a delocalized positive charge in the adenine moiety that promotes stretching of the N‐ribosidic bond to progress the catalytic reaction. Currently, transition state analogs are known to function as powerful inhibitors of MTAN; however, the majority of these compounds exhibit inhibition of the human enzyme 5′‐methylthioadenosine phosphorylase (hMTAP), a homolog to HpMTAN. In this study, we present the first neutron structures of an active and an inactive variant (D198N) of HpMTAN that were co‐crystallized with SAH. The structures were solved using X‐ray crystallography and subjected to joint X‐ray/neutron refinement to allow for direct determination of hydrogen atom positioning throughout the catalytic domain of HpMTAN. The neutron structures confirm the proposed mechanism in which D198 acts as a general acid and illustrates other features of the catalytic reaction. Additionally, key polar interactions are highlighted in the catalytic domain that will aid in structure‐based design of inhibitors that will be selective for HpMTAN and have little to no effect on hMTAP.