Crystal Structures of the Helicobacter pylori MTAN Enzyme Reveal Specific Interactions between S-Adenosylhomocysteine and the 5′-Alkylthio Binding Subsite

Abstract

The bacterial 5’-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) enzyme is a multifunctional enzyme that catalyzes the hydrolysis of the N-ribosidic bond of at least four different adenosine-based metabolites: S-adenosylhomocysteine (SAH), 5’methylthioadenosine (MTA), 5’-deoxyadenosine (5’-DOA) and 6-amino-6-deoxyfutalosine. These activities place the enzyme at the hub of seven fundamental bacterial metabolic pathways: S-adenosylmethionine (SAM) utilization, polyamine biosynthesis, the purine salvage pathway, the methionine salvage pathway, the SAM radical pathways, autoinducer-2 biosynthesis, and menaquinone biosynthesis. The last pathway makes MTAN essential for H. pylori viability. Although structures of various bacterial and plant MTANs have been described, the interactions between the homocysteine moiety of SAH and the 5’-alkylthiol binding site of MTAN have never been resolved. We have solved crystal structures of an inactive mutant form of Helicobacter pylori MTAN bound to MTA and SAH to 1.63 and 1.20 Å, respectively. The active form of MTAN was also crystallized in the presence of SAH allowing structure determination of a ternary enzyme-product complex resolved at 1.50 Å. These structures identify interactions between the homocysteine moiety and the 5’-alkylthiol binding site of the enzyme. This information can be leveraged for the development of species-specific MTAN inhibitors that prevent the growth of H. pylori.

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

“…The other δND 2 atom of N198 forms an N-D···N hydrogen bond with N7 of the SAH at a distance of 2.1 Å. In the previously published HpMTAN-D198N X-ray crystal structures, this pocket always contains a single ordered water molecule, but no corresponding density is observed in the wild-type structures (5,12,15). Although ordering of this water molecule could simply be a consequence of the D198N mutation, it is expected that this pocket contains a disordered water molecule in the wild-type enzyme.…”

“…Indeed, assessment of side-chain ionization of the analogous residue in the Escherichia coli homolog determined a pK a of 8.2, which can be attributed to the burial of the D198 side chain upon substrate binding (9,14). Additionally, D198 has been demonstrated to be essential for the enzymatic activity through the use of an asparagine variant (D198N) of HpMTAN that binds substrate but does not promote hydrolysis (5,12,15). Following protonation of the substrate by D198, the adenine leaving group becomes electron withdrawing, leading to elongation of the N-ribosidic bond.…”

Structural and Biochemical Characterization of Chlamydia trachomatis Hypothetical Protein CT263 Supports That Menaquinone Synthesis Occurs through the Futalosine Pathway

“…4B). The presence of a single occupied active site in substrate-bound complexes, as described within the ligand-bound CT263 structures, is quite common in proto- typical MTANs (52,54,59). The recent structure of ligandbound SeMTAN highlights this effect, where one monomer was in the "closed" form, and the other remained empty and in the "open" form (54).…”

“…This region is typically involved in dimerization (Fig. 7), where it contributes side chains involved in active site interactions within all structurally characterized MTANs (41,(52)(53)(54)(55)(56)(57)(58). The absence of this region reflects the altered CT263 dimer assemblies relative to the prototypical MTAN dimer interface.…”

“…Structural Comparison with SeMTAN Reveals That CT263 Is in the Closed Form-MTANs have been extensively studied from a structure/function standpoint in a variety of medically relevant pathogens, including E. coli (41,56,58), H. pylori (52,53,57,59), S. aureus (60), and S. enterica (54), among others. All of these structures have crystallized with a conserved dimer interface (Fig.…”