Neutron structures of the Helicobacter pylori 5′-methylthioadenosine nucleosidase highlight proton sharing and protonation states

Abstract

MTAN (5′-methylthioadenosine nucleosidase) catalyzes the hydrolysis of the N-ribosidic bond of a variety of adenosine-containing metabolites. The Helicobacter pylori MTAN (HpMTAN) hydrolyzes 6-amino-6-deoxyfutalosine in the second step of the alternative menaquinone biosynthetic pathway. Substrate binding of the adenine moiety is mediated almost exclusively by hydrogen bonds, and the proposed catalytic mechanism requires multiple protontransfer events. Of particular interest is the protonation state of residue D198, which possesses a pK a above 8 and functions as a general acid to initiate the enzymatic reaction. In this study we present three corefined neutron/X-ray crystal structures of wildtype HpMTAN cocrystallized with S-adenosylhomocysteine (SAH), Formycin A (FMA), and (3R,4S)-4-(4-Chlorophenylthiomethyl)-1-[(9-deazaadenin-9-yl)methyl]-3-hydroxypyrrolidine (p-ClPh-Thio-DADMe-ImmA) as well as one neutron/X-ray crystal structure of an inactive variant (HpMTAN-D198N) cocrystallized with SAH. These results support a mechanism of D198 pKa elevation through the unexpected sharing of a proton with atom N7 of the adenine moiety possessing unconventional hydrogen-bond geometry. Additionally, the neutron structures also highlight active site features that promote the stabilization of the transition state and slight variations in these interactions that result in 100-fold difference in binding affinities between the DADMe-ImmA and ImmA analogs.neutron diffraction | enzyme mechanism | proton transfer | nucleosidase | Helicobacter

Neutron crystallography of copper amine oxidase reveals keto/enolate interconversion of the quinone cofactor and unusual proton sharing

“…Hence, it is assumed that the strong interaction of the triply shared proton is responsible for the force that bends the quinone ring, albeit slightly, as described below. Similar triply shared protons have been observed in the neutron structure of Helicobacter pylori 5′-methylthioadenosine nucleosidase (11).…”

Unusual zwitterionic catalytic site of SARS–CoV-2 main protease revealed by neutron crystallography

“…Because D atoms scatter neutrons just as well as other protein atoms, they can be directly detected in neutron structures at moderate resolutions as low as 2.5-2.6 Å. 38,39 Notably, sulfur (S) has a coherent neutron scattering length of +2.847 fm, less than half the magnitude of that for carbon, oxygen, nitrogen, and deuterium. Consequently, deprotonated thiol groups (S -) in cysteine (Cys) and side chain S atoms in methionine (Met) residues are often not easily visible in nuclear density maps.…”

Protonation states in SARS-CoV-2 main protease mapped by neutron crystallography

“…Because D atoms scatter neutrons just as well as other protein atoms, they can be directly detected in neutron structures at moderate resolutions as low as 2.5-2.6 Å. 38,39 Notably, sulfur (S) has a coherent neutron scattering length of +2.847 fm, less than half the magnitude of that for carbon, oxygen, nitrogen, and deuterium. Consequently, deprotonated thiol groups (S -) in cysteine (Cys) and side chain S atoms in methionine (Met) residues are often not visible in nuclear density maps.…”