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 Å 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.

Introduction

Methionine-recycling pathways have evolved in eukaryotes and most bacterial species because the amino acid is often limited in bioavailability and is energetically costly to synthesize de novo.1, 2 Methionine is essential for cellular metabolism and development, and accretion of the nucleoside metabolites, 5′-methylthioadenosine (MTA) and S-adenosylhomocysteine (SAH) (Fig. 1), negatively regulates transmethylation reactions and quorum sensing, and polyamine biosynthesis, respectively.3, 4 In mammals, MTA is converted to 5-methylthioribose 1-phosphate (MTR 1-P) and adenine by MTA phosphorylase (MTAP),⁵ while SAH is metabolized to homocysteine and adenosine by SAH hydrolase. MTAP is not found in plants and many bacterial species. In its place, MTA nucleosidase (MTAN) is used to hydrolyze MTA, and in some cases SAH. MTAN catalyzes the irreversible cleavage of the N-glycosidic bond of MTA and SAH to form adenine, and methylthioribose (MTR) and S-ribosylhomocysteine (SRH), respectively.6, 7, 8, 9, 10 Adenine enters the nucleotide biosynthetic pathway, while MTR, depending on the species, is either excreted or phosphorylated at the C1-hydroxyl by MTR kinase to form MTR 1-P. MTR 1-P is subsequently isomerized to 5-methylthioribulose 1-phosphate by MTR 1-P epimerase.¹¹ The ensuing downstream steps in the methionine recycling pathway appear to vary somewhat between species, depending largely on whether the environment is aerobic or anaerobic, and this has been reviewed in detail by Sekowska et al. ¹¹ The last step is common to all species and involves an amino group transfer from a number of potential amino acid donors to 2-keto-4-methylthiobutyrate to form methionine.¹¹

While bacterial MTANs show dual-substrate specificity for both MTA and SAH, plant MTANs (typically referred to in the plant literature as MTN) exhibit a distinct preference for MTA with little or no activity towards SAH.12, 13, 14 Like mammals, plants appear to utilize SAH hydrolase to metabolize SAH.15, 16 In addition, the methionine-recycling pathway in plants is conjugated to ethylene synthesis.¹⁷ In the first step of ethylene synthesis, 1-aminocyclopropane-1-carboxylate (ACC) is produced from S-adenosylmethionine (AdoMet) with MTA released as a side product.¹⁸ Accumulation of MTA inhibits ethylene production.17, 19 Regulation of the methionine recycling pathway by ethylene is observed in plants that synthesize high levels of ethylene for prolonged periods.¹⁷ These include plants with seasonal fruit ripening, such as tomatoes, and those that are submerged in water for extended periods, such as rice. In these species, MTAN is suggested to be important in seed germination and fruit ripening.¹⁶

The metabolic variation between the bacterial and plant MTAN's suggests structural differences that affect substrate recognition and binding. The structures and mutational analysis of a representative bacterial MTAN, Escherichia coli MTAN (EcMTAN), have identified the catalytic residues,20, 21, 22 and kinetic isotope effect studies²³ support the proposed S_N1-type catalytic mechanism, which is thought to begin with the donation of a proton from the catalytic acid (Asp197) to N₇ of the substrate. The protonated adenine base withdraws electrons from the ribose group, leading to elongation of the ribosidic bond and the development of a partial positive charge on the ribose group that is stabilized by the catalytic water molecule (WAT) and Glu174. Glu12 abstracts a proton from the catalytic water molecule, which then attacks C_1′ of the oxocarbenium-like intermediate to form the products.

In contrast, structural and kinetic studies on the plant enzyme(s) are more limited. The first structure of a representative plant MTAN from Arabidopsis thaliana (AtMTAN1) was determined recently in complex with the product, adenine (PDB code 2H8G).²⁴ To better understand the mechanisms of substrate-recognition and catalysis, we have examined the substrate specificities and activity of the two MTAN homologues present in A. thaliana, and have determined the structure of AtMTAN1 in its apo form, and in complex with the substrate-analogue, 5′-methylthiotubercidin (MTT), and the transition-state analogue, formycin A (FMA) (Fig. 1). A structural homology model of AtMTAN2 has been constructed, which highlights differences in the active sites of the two isozymes that are likely critical for the substrate specificity observed. Analysis of the ligand-bound structures of EcMTAN and AtMTAN1 further suggest that differences in the dynamics, size and specificity of the active sites may account for the divergence in substrate selectivity between the bacterial and plant enzymes.