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Structure of nicotinic acid mononucleotide adenylyltransferase from Bacillus anthracis.

ABSTRACT: Nicotinic acid mononucleotide adenylyltransferase (NaMNAT; EC 2.7.7.18) is the penultimate enzyme in the biosynthesis of NAD(+) and catalyzes the adenylation of nicotinic acid mononucleotide (NaMN) by ATP to form nicotinic acid adenine dinucleotide (NaAD). This enzyme is regarded as a suitable candidate for antibacterial drug development; as such, Bacillus anthracis NaMNAT (BA NaMNAT) was heterologously expressed in Escherichia coli for the purpose of inhibitor discovery and crystallography. The crystal structure of BA NaMNAT was determined by molecular replacement, revealing two dimers per asymmetric unit, and was refined to an R factor and R(free) of 0.228 and 0.263, respectively, at 2.3 A resolution. The structure is very similar to that of B. subtilis NaMNAT (BS NaMNAT), which is also a dimer, and another independently solved structure of BA NaMNAT recently released from the PDB along with two ligated forms. Comparison of these and other less related bacterial NaMNAT structures support the presence of considerable conformational heterogeneity and flexibility in three loops surrounding the substrate-binding area.

SUBMITTER: Lu S

PROVIDER: S-EPMC2564882 | biostudies-literature | 2008 Oct

REPOSITORIES: biostudies-literature

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Structure of nicotinic acid mononucleotide adenylyltransferase from Bacillus anthracis.

Lu Shanyun S Smith Craig D CD Yang Zhengrong Z Pruett Pamela S PS Nagy Lisa L McCombs Deborah D Delucas Lawrence J LJ Brouillette Wayne J WJ Brouillette Christie G CG

Acta crystallographica. Section F, Structural biology and crystallization communications 20080930 Pt 10

Nicotinic acid mononucleotide adenylyltransferase (NaMNAT; EC 2.7.7.18) is the penultimate enzyme in the biosynthesis of NAD(+) and catalyzes the adenylation of nicotinic acid mononucleotide (NaMN) by ATP to form nicotinic acid adenine dinucleotide (NaAD). This enzyme is regarded as a suitable candidate for antibacterial drug development; as such, Bacillus anthracis NaMNAT (BA NaMNAT) was heterologously expressed in Escherichia coli for the purpose of inhibitor discovery and crystallography. The ...[more]

PMID: 18931430

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Project description:NAD and its reduced form NADH function as essential redox cofactors and have major roles in determining cellular metabolic features. NAD can be synthesized through the deamidated and amidated pathways, for which the key reaction involves adenylylation of nicotinic acid mononucleotide (NaMN) and nicotinamide mononucleotide (NMN), respectively. In Escherichia coli, NAD de novo biosynthesis depends on the protein NadD-catalyzed adenylylation of NaMN to nicotinic acid adenine dinucleotide (NaAD), followed by NAD synthase-catalyzed amidation. In this study, we engineered NadD to favor NMN for improved amidated pathway activity. We designed NadD mutant libraries, screened by a malic enzyme-coupled colorimetric assay, and identified two variants, 11B4 (Y84V/Y118D) and 16D8 (A86W/Y118N), with a high preference for NMN. Whereas in the presence of NMN both variants were capable of enabling the viability of cells of E. coli BW25113-derived NAD-auxotrophic strain YJE003, for which the last step of the deamidated pathway is blocked, the 16D8 expression strain could grow without exogenous NMN and accumulated a higher cellular NAD(H) level than BW25113 in the stationary phase. These mutants established fully active amidated NAD biosynthesis and offered a new opportunity to manipulate NAD metabolism for biocatalysis and metabolic engineering.IMPORTANCE Adenylylation of nicotinic acid mononucleotide (NaMN) and adenylylation of nicotinamide mononucleotide (NMN), respectively, are the key steps in the deamidated and amidated pathways for NAD biosynthesis. In most organisms, canonical NAD biosynthesis follows the deamidated pathway. Here we engineered Escherichia coli NaMN adenylyltransferase to favor NMN and expressed the mutant enzyme in an NAD-auxotrophic E. coli strain that has the last step of the deamidated pathway blocked. The engineered strain survived in M9 medium, which indicated the implementation of a functional amidated pathway for NAD biosynthesis. These results enrich our understanding of NAD biosynthesis and are valuable for manipulation of NAD homeostasis for metabolic engineering.

Dataset Information

Structure of nicotinic acid mononucleotide adenylyltransferase from Bacillus anthracis.

Publications

Structure of nicotinic acid mononucleotide adenylyltransferase from Bacillus anthracis.

Similar Datasets

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