Kinetic and X-Ray Structural Evidence for Negative Cooperativity in Substrate Binding to Nicotinate Mononucleotide Adenylyltransferase (NMAT) from Bacillus anthracis

Abstract

Biosynthesis of NAD(P) in bacteria occurs through either the de novo or one of the salvage pathways which converge at the point where the reaction of nicotinate mononucleotide (NaMN) with adenosine triphosphate (ATP), is coupled to the formation of nicotinate adenine dinucleotide (NaAD) and inorganic pyrophosphate (PP i ). This reaction is catalyzed by nicotinate mononucleotide adenylyltransferase (NMAT) which is essential for bacterial growth making it an attractive drug target for the development of new antibiotics. Steady-state kinetic and direct binding studies on NMAT from Bacillus anthracis suggest a random sequential Bi-Bi kinetic mechanism. Interestingly, the interactions of NaMN and ATP with NMAT were observed to exhibit negative cooperativity, i.e. Hill coefficients < 1.0. Negative cooperativity in binding is also supported from X-ray crystallographic studies. X-ray structures of the B. anthracis NMAT apoenzyme, and the NaMN and NaAD-bound complexes were determined to resolutions of 2.50 Å, 2.60 Å and 1.75 Å, respectively. The X-ray structure of the NMAT-NaMN complex revealed only one NaMN molecule bound in the biological dimer supporting negative cooperativity in substrate binding. The kinetic, direct-binding, and X-ray structural studies support a model whereby the binding affinity of substrates to the first monomer of NMAT is stronger than to the second and analysis of the three X-ray structures reveals significant conformational changes of NMAT along the enzymatic reaction coordinate. The negative cooperativity observed in B. anthracis NMAT substrate binding is a unique property that has so far not yet been observed in other prokaryotic NMAT enzymes. We propose that regulation of the NAD (P) biosynthetic pathway may in part occur at the reaction catalyzed by NMAT.

Mycobacterial Nicotinate Mononucleotide Adenylyltransferase

“…5) that has been reported to act as an arm to recognize the NaMN substrate or, more importantly, to bind the NaAD product. This loop, usually disordered in the apo form of bacterial NadDs, becomes ordered upon contact with the substrate or product (22,23,37,38). Because this loop was not fully resolved in our structure, which is not uncommon in NadD structure apo forms, and to better elucidate its mechanistic role, we carried out a full alanine-scanning mutagenesis of the partially conserved motif Pro-44 -Lys-47.…”

“…However, bacterial NadD enzymes have been less studied, and although the three-dimensional structures were reported for some bacterial pathogens (16,(22)(23)(24), it has not been reported for NadD from Mtb. Recently, we reported for the first time the successful expression, purification, and basic kinetic properties of MtNadD (17).…”

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.…”

“…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.…”

“…2). In the case of bacterial NMNATs, the structural comparison of substratebound [17] and product-bound [16,[18][19][20][21] forms reveals that pronounced conformational changes occur only upon binding of both substrates ATP and NaMN. Similar structural rearrangements are also observed in human and archaeal NMNTAs, although to a smaller extent [22][23][24][25].…”