Mechanism of substrate specificity in 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidases

Abstract

5′-Methylthioadenosine/S-adenosylhomocysteine (MTA/SAH) nucleosidase (MTAN) plays a key role in the methionine-recycling pathway of bacteria and plants. Despite extensive structural and biochemical studies, the molecular mechanism of substrate specificity for MTAN remains an outstanding question. Bacterial MTANs show comparable efficiency in hydrolyzing MTA and SAH, while the plant enzymes select preferentially for MTA, with either no or significantly reduced activity towards SAH. Bacterial and plant MTANs show significant conservation in the overall structure, and the adenine- and ribose-binding sites. The observation of a more constricted 5′-alkylthio binding site in Arabidopsis thaliana AtM-TAN1 and AtMTAN2, two plant MTAN homologues, led to the hypothesis that steric hindrance may play a role in substrate selection in plant MTANs. We show using isothermal titration calorimetry that SAH binds to both Escherichia coli MTAN (EcMTAN) and AtMTAN1 with comparable micromolar affinity. To understand why AtMTAN1 can bind but not hydrolyze SAH, we determined the structure of the protein–SAH complex at 2.2 Å resolution. The lack of catalytic activity appears to be related to the enzyme’s inability to bind the substrate in a catalytically competent manner. The role of dynamics in substrate selection was also examined by probing the amide proton exchange rates of EcMTAN and AtMTAN1 via deuterium–hydrogen exchange coupled mass spectrometry. These results correlate with the B factors of available structures and the thermodynamic parameters associated with substrate binding, and suggest a higher level of conformational flexibility in the active site of EcMTAN. Our results implicate dynamics as an important factor in substrate selection in MTAN.

Cofactome analyses reveal enhanced flux of carbon into oil for potential biofuel production

“…The methionine‐recycling pathway in Matilda is also distinct from that of Freja, as evidenced by higher expression levels of the gene encoding 5′‐methylthioadenosine/ S ‐adenosylhomocysteine nucleosidase (MTAN), a methionine‐recycling enzyme (Siu et al. , 2011), as well as higher steady‐state levels of the respective product deoxyadenosine.…”

Pfs promotes autolysis-dependent release of eDNA and biofilm formation in Staphylococcus aureus

“…Until recently, studies concerning Pfs have primarily focused on determining the biochemical properties of this enzyme [41], screening the Pfs inhibitors for the study of their putative antimicrobial activity [42] or identifying the role of Pfs related to the AI-2 signal. In S. aureus, the structure of Pfs and the MTA and SAH nucleosidase activity of this enzyme have been determined [43].…”

Recycling of Methylthioadenosine Is Essential for Normal Vascular Development and Reproduction in Arabidopsis

“…Thus, it was initially inferred that these two enzymes may have distinct roles in plant metabolism: in vitro, MTN1 accepts only MTA as a substrate, while MTN2 can also accept S-adenosylhomocysteine to a limited extent (Siu et al, 2008). More recent crystallography and protein dynamic analyses revealed that MTN1 binds to S-adenosylhomocysteine but is incapable of hydrolyzing it (Siu et al, 2011).…”