Molecular Determinants of Substrate Specificity in Plant 5′-Methylthioadenosine Nucleosidases

Abstract

5'-Methylthioadenosine (MTA)/S-adenosylhomocysteine (SAH) nucleosidase (MTAN) is essential for cellular metabolism and development in many bacterial species. While the enzyme is found in plants, plant MTANs appear to select for MTA preferentially, with little or no affinity for SAH. To understand what determines substrate specificity in this enzyme, MTAN homologues from Arabidopsis thaliana (AtMTAN1 and AtMTAN2, which are referred to as AtMTN1 and AtMTN2 in the plant literature) have been characterized kinetically. While both homologues hydrolyze MTA with comparable kinetic parameters, only AtMTAN2 shows activity towards SAH. AtMTAN2 also has higher catalytic activity towards other substrate analogues with longer 5'-substituents. The structures of apo AtMTAN1 and its complexes with the substrate- and transition-state-analogues, 5'-methylthiotubercidin and formycin A, respectively, have been determined at 2.0-1.8 A resolution. A homology model of AtMTAN2 was generated using the AtMTAN1 structures. Comparison of the AtMTAN1 and AtMTAN2 structures reveals that only three residues in the active site differ between the two enzymes. Our analysis suggests that two of these residues, Leu181/Met168 and Phe148/Leu135 in AtMTAN1/AtMTAN2, likely account for the divergence in specificity of the enzymes. Comparison of the AtMTAN1 and available Escherichia coli MTAN (EcMTAN) structures suggests that a combination of differences in the 5'-alkylthio binding region and reduced conformational flexibility in the AtMTAN1 active site likely contribute to its reduced efficiency in binding substrate analogues with longer 5'-substituents. In addition, in contrast to EcMTAN, the active site of AtMTAN1 remains solvated in its ligand-bound forms. As the apparent pK(a) of an amino acid depends on its local environment, the putative catalytic acid Asp225 in AtMTAN1 may not be protonated at physiological pH and this suggests the transition state of AtMTAN1, like human MTA phosphorylase and Streptococcus pneumoniae MTAN, may be different from that found in EcMTAN.

Pseudomonas syringae type III effector HopAF1 suppresses plant immunity by targeting methionine recycling to block ethylene induction

“…MTN1 N113 is involved in ligand binding via coordination of a water molecule in the ligand-binding pocket (Fig. 5A) (71). MTN1 N194 and MTN1 N169 are both surface exposed at the end of helices α4 and α3, respectively (Fig.…”

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

“…Recently, it has also been shown that both MTNs are abundant in the phloem tissue (Pommerrenig et al, 2011). Although MTN1 and MTN2 polypeptides share 64% amino acid sequence identity, they have distinct substrate specificities and pH optima (Siu et al, 2008). 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).…”

“…Although MTN1 and MTN2 polypeptides share 64% amino acid sequence identity, they have distinct substrate specificities and pH optima (Siu et al, 2008). 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).…”

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

“…Many plant MTAN homologues are unable to metabolize SAH and those that do show 84–87% reduction in activity relative to the hydrolysis of MTA (Guranowski et al, 1981; Rzewuski et al, 2007; Baxter and Coscia, 1973). Kinetic characterization of the two MTAN homologues found in the thale cress plant, Arabidopsis thaliana, showed that At MTAN1 is completely inactive towards SAH while At MTAN2 has 14% activity towards this substrate (Siu et al, 2008a). Structural comparison of At MTAN1 with the bacterial MTAN homologue from Escherichia coli ( Ec M-TAN) showed significant conservation in the overall structure (Siu et al, 2008a).…”

“…Kinetic characterization of the two MTAN homologues found in the thale cress plant, Arabidopsis thaliana, showed that At MTAN1 is completely inactive towards SAH while At MTAN2 has 14% activity towards this substrate (Siu et al, 2008a). Structural comparison of At MTAN1 with the bacterial MTAN homologue from Escherichia coli ( Ec M-TAN) showed significant conservation in the overall structure (Siu et al, 2008a). Close examination of the active sites suggested that the active site of Ec MTAN may be more disordered than that of At MTAN1 (Siu et al, 2008a).…”