Mutational, Structural, and Kinetic Studies of the ATP-binding Site of Methanobacterium thermoautotrophicum Nicotinamide Mononucleotide Adenylyltransferase

Abstract

Several residues lining the ATP-binding site of Methanobacterium thermoautotrophicum nicotinamide mononucleotide adenylyltransferase (NMNATase) were mutated in an effort to better characterize their roles in substrate binding and catalysis. Residues selected were Arg-11 and Arg-136, both of which had previously been implicated as substrate binding residues, as well as His-16 and His-19, part of the HXGH active site motif and postulated to be of importance in catalysis. Kinetic studies revealed that both Arg-11 and Arg-136 contributed to the binding of the substrate, ATP. When these amino acids were replaced by lysines, the apparent K m values of the respective mutants for ATP decreased by factors of 1.3 and 2.9 and by factors of 1.9 and 8.8 when the same residues were changed to alanines. ϩ served as substrates with quite different affinities (3, 6). Genomic analysis of E. coli revealed, however, that in this organism there are two homologous but distinct genes, NadD and NadR, which encode the enzymes nicotinic acid mononucleotide adenylyltransferase and nicotinamide mononucleotide adenylyltransferase, respectively (7,8). Searching the M. thermoautotrophicum proteomic data base for homologues of NadD, however, did not identify any putative nicotinic acid mononucleotide adenylyltransferase. Archaeal proteomes seem to contain only a single protein that catalyzes both activities. All archaeal enzymes contain an invariant glutamine residue at position 84 that binds to the amide nitrogen of the NMN ϩ substrate via its amide oxygen side chain (9). A possible model, which would explain the dual enzymatic activity, has free rotation around the glutamine side chain allowing its amide nitrogen to bind to the carboxylate of NaMN ϩ making it quite feasible for a single enzyme to catalyze both reactions.Recently, there has been increased interest in the biochemistry of NMNATases; the genes encoding these enzymes in several bacterial, archaeal, and eukaryotic sources including humans have been identified (3, 5, 6, 8 -14). The structures of M. thermoautotrophicum and M. jannaschii NMNATase were determined in complex with the product NAD ϩ or the substrate ATP, respectively (9, 15). More recently, crystal structures were reported for the apo-form of human NMNATase (16) and its complexes with NMN ϩ (17), NAD ϩ (18), NaAD ϩ (18), and the oncolytic agent tiazofurin (18). All these analyses revealed a hexameric assembly and a highly similar overall fold. The structures of Bacillus subtilis and E. coli nicotinic acid mononucleotide adenylyltransferase (the products of the NadD gene) were also determined (19,20). In contrast to the archaeal and This work was supported by a grant from the Natural Sciences and Engineering Council, Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.The

A Novel NAD-binding Protein Revealed by the Crystal Structure of 2,3-Diketo-l-gulonate Reductase (YiaK)

“…This conformational difference is because of a 140°rota-tion around the bond linking the ␣-phosphate and the bridging oxygen atom, as well as a rotation of 120°around the C-4Ј-C-5Ј bond of the ribose. Interestingly, this NAD conformation has recently been observed in a complex with a mutant of nicotinamide-mononucleotide adenylyltransferase (20). The conformation of NAD in complex with the wild type of this enzyme is, however, entirely different (Fig.…”

Nicotinamide/nicotinic acid mononucleotide adenylyltransferase, new insights into an ancient enzyme

“…Indeed, the structures of NMNAT from archaea [13][14][15], eubacteria [16][17][18][19][20][21], and eukarya [22][23][24][25] have been reported. Interestingly, the NMNAT enzymatic activity is also found in proteins endowed with dual functions, whereby the NAD biosynthetic activity is associated with a second function residing on a different domain in a chimeric protein.…”

“…1 and 3). As observed in the structure of the ATP complex with the prototypical MjNMNAT [13], the first histidine in the (H/T)XXH motif interacts with the ATP b-phosphate whereas the second conserved histidine in the signature binds to the a-phosphate and is an essential residue for catalysis as demonstrated by site-directed mutagenesis experiments in both archaeal and human enzymes [15,37]. domain to the C-terminal domain is depicted in orange.…”

“…Therefore, it could be argued that the NMN versus NaMN binding sites in enzymes from different sources should diverge and contain peculiar structural determinants for the recognition of either form of the substrate. However, although the structures of several NMNATs in complex with NMN, NaMN, NAD or NaAD [14][15][16][17][18][19][20][21][23][24][25] have been reported, no exhaustive and general answer can be provided to the question of which and to what extent conserved versus peculiar structural determinants contribute to pyridine mononucleotide binding and dictate NMN versus NaMN substrate selectivity in the different enzymes. Even though satisfactory explanations have been provided for specific enzymes, no conserved structural patterns responsible for pyridine mononucleotide selectivity have emerged from the wealth of available structural data.…”

“…The enzyme active site appears to be carefully designed to accommodate and properly orient the reactant groups without direct involvement of protein residues in covalent or acid/base catalysis [6,13]. The role of the protein milieu is also essential for stabilization of the transition state, with the second histidine of the (H/T)XXH signature likely being a key residue in this respect [13,15,37] (Fig. 3).…”