Early Evolutionary Selection of NAD Biosynthesis Pathway in Bacteria

Abstract

Bacteria use two alternative pathways to synthesize nicotinamide adenine dinucleotide (NAD) from nicotinamide (Nam). A short, two-step route proceeds through nicotinamide mononucleotide (NMN) formation, whereas the other pathway, a four-step route, includes the deamidation of Nam and the reamidation of nicotinic acid adenine dinucleotide (NAAD) to NAD. In addition to having twice as many enzymatic steps, the four-step route appears energetically unfavourable, because the amidation of NAAD includes the cleavage of ATP to AMP. Therefore, it is surprising that this pathway is prevalent not only in bacteria but also in yeast and plants. Here, we demonstrate that the considerably higher chemical stability of the deamidated intermediates, compared with their amidated counterparts, might compensate for the additional energy expenditure, at least at elevated temperatures. Moreover, comprehensive bioinformatics analyses of the available >6000 bacterial genomes indicate that an early selection of one or the other pathway occurred. The mathematical modelling of the NAD pathway dynamics supports this hypothesis, as there appear to be no advantages in having both pathways.

Engineering the C3N Pathway as a Short Detour for De Novo NAD+ Biosynthesis in Saccharomyces cerevisiae

“…NAD + is a life-essential metabolite involved in numerous redox reactions and multiple cellular physiological processes. Since it was discovered as the first 'cozymase' more than 100 years ago, two natural de novo NAD + biosynthetic pathways (pathway I and II) have been elucidated and both of them start from a proteinogenic amino acid [4,9]. In a previous study, we designed an alternative de novo NAD + biosynthesis pathway, the C3N pathway that starts from chorismate, and established this pathway successfully in E. coli by expressing four exogenous genes encoding an ADIC synthase, a DHHA synthase, a DHHA dehydrogenase, and a 3-HAA 3,4-dioxygenase [16].…”

“…In a previous study, we designed an alternative de novo NAD + biosynthesis pathway, the C3N pathway that starts from chorismate, and established this pathway successfully in E. coli by expressing four exogenous genes encoding an ADIC synthase, a DHHA synthase, a DHHA dehydrogenase, and a 3-HAA 3,4-dioxygenase [16]. Chorismate can be converted to 3-HAA by the former three enzymes, and then undergo the oxidative ring cleavage catalyzed by 3-HAA 3,4-dioxygenase and a spontaneous cyclization to form QA, which will enter the native de novo NAD + biosynthetic pathway I of E. coli and generate NAD + via three steps common to all de novo NAD + biosynthetic pathways [9,16]. Our previous work demonstrated the advantages of the C3N pathway in prokaryotic cells.…”

“…To date, only two natural de novo NAD + biosynthesis pathways have been delineated [4,9]. Pathway I, which distributes in plants and most bacteria, uses Laspartate (L-Asp) as a precursor for the nicotinamide moiety of NAD + [10,11], whereas pathway II, which exists in some bacteria, mammals, and fungi, recruits L-tryptophan (L-Trp) as a precursor [4,12].…”

“…Subsequently, 3-HAA is converted to quinolinic acid (QA) by 3-HAA 3,4-dioxygenase, which is also a key enzyme in pathway II for NAD + synthesis [17]. The three steps from QA to NAD + are conserved for all de novo biosynthesis pathways of NAD + [9]. The newly developed C3N pathway circumvents the stringent regulation on de novo NAD + biosynthesis and results in a more than nine times improvement of the cellular NAD(H) level in E. coli, demonstrating its powerfulness in cofactor engineering [16].…”