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Evidence that the nadA motif is a bacterial riboswitch for the ubiquitous enzyme cofactor NAD.

ABSTRACT: The nadA motif is a riboswitch candidate present in various Acidobacteria species that was previously identified by bioinformatic analysis of bacterial DNA data sets. More than 100 unique representatives have been identified exclusively upstream of nadA genes, which code for an enzyme in the biosynthetic pathway of the ubiquitous coenzyme NAD+ The architecture of nadA motif RNAs suggests they use structurally similar tandem ligand-binding aptamer domains to control translation initiation. Biochemical analyses reveal that the first domain selectively binds ligands carrying an adenosine 5'-diphosphate (5' ADP) moiety, including NAD+ and its reduced form, NADH. Genetic analyses indicate that a tandem nadA motif RNA suppresses gene expression when NAD+ is abundant, and that both aptamer domains are required for maximal gene regulation. However, we have not observed selective binding of the nicotinamide moiety of NAD+ or binding by the second putative aptamer in vitro, despite sequence and structural similarities between the tandem domains.

SUBMITTER: Malkowski SN

PROVIDER: S-EPMC6859854 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Evidence that the nadA motif is a bacterial riboswitch for the ubiquitous enzyme cofactor NAD.

Malkowski Sarah N SN Spencer Tara C J TCJ Breaker Ronald R RR

RNA (New York, N.Y.) 20190829 12

The nadA motif is a riboswitch candidate present in various Acidobacteria species that was previously identified by bioinformatic analysis of bacterial DNA data sets. More than 100 unique representatives have been identified exclusively upstream of nadA genes, which code for an enzyme in the biosynthetic pathway of the ubiquitous coenzyme NAD+ The architecture of nadA motif RNAs suggests they use structurally similar tandem ligand-binding aptamer domains to contro ...[more]

PMID: 31467147

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Project description:BackgroundCofactor engineering is involved in the modification of enzymes related to nicotinamide adenine dinucleotides (NADH and NAD+) metabolism, which results in a significantly altered spectrum of metabolic products. Cofactor engineering plays an important role in metabolic engineering but is rarely reported in the sterols biotransformation process owing to its use of multi-catabolic enzymes, which promote multiple consecutive reactions. Androst-4-ene-3, 17-dione (AD) and androst-1, 4-diene-3, 17-dione (ADD) are important steroid medicine intermediates that are obtained via the nucleus oxidation and the side chain degradation of phytosterols by Mycobacterium. Given that the biotransformation from phytosterols to AD (D) is supposed to be a NAD+-dependent process, this work utilized cofactor engineering in Mycobacterium neoaurum and investigated the effect on cofactor and phytosterols metabolism.ResultsThrough the addition of the coenzyme precursor of nicotinic acid in the phytosterols fermentation system, the intracellular NAD+/NADH ratio and the AD (D) production of M. neoaurum TCCC 11978 (MNR M3) were higher than in the control. Moreover, the NADH: flavin oxidoreductase was identified and was supposed to exert a positive effect on cofactor regulation and phytosterols metabolism pathways via comparative proteomic profiling of MNR cultured with and without phytosterols. In addition, the NADH: flavin oxidoreductase and a water-forming NADH oxidase from Lactobacillus brevis, were successfully overexpressed and heterologously expressed in MNR M3 to improve the intracellular ratio of NAD+/NADH. After 96 h of cultivation, the expression of these two enzymes in MNR M3 resulted in the decrease in intracellular NADH level (by 51 and 67%, respectively) and the increase in NAD+/NADH ratio (by 113 and 192%, respectively). Phytosterols bioconversion revealed that the conversion ratio of engineered stains was ultimately improved by 58 and 147%, respectively. The highest AD (D) conversion ratio by MNR M3N2 was 94% in the conversion system with soybean oil as reaction media to promote the solubility of phytosterols.ConclusionsThe ratio of NAD+/NADH is an important factor for the transformation of phytosterols. Expression of NADH: flavin oxidoreductase and water-forming NADH oxidase in MNR improved AD (D) production. Besides the manipulation of key enzyme activities, which included in phytosterols degradation pathways, maintenance the balance of redox also played an important role in promoting steroid biotransformation. The recombinant MNR strain may be useful in industrial production.

Dataset Information

Evidence that the nadA motif is a bacterial riboswitch for the ubiquitous enzyme cofactor NAD.

Publications

Evidence that the <i>nadA</i> motif is a bacterial riboswitch for the ubiquitous enzyme cofactor NAD<sup>.</sup>

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