NAD P Biosynthesis

Nicotinamide serves as the major precursor of NAD in mammalians. The reaction with phosphoribosyl pyrophosphate (PRPP) leads to the formation of nicodtiamide mononucleotide, NMN. Finally, this mononucleotide is linked to the adenylate moiety of ATP forming NAD (Fig. 2). This reaction is catalysed by nicotinamide mononucleotide adenylyltransferases, NMNATs. 

As an alternative to nicotinamide, quinolinic acid (a degradation product of tryptophan) may be used to form nicotinic acid mononucleotide (NaMN). Quinolinic acid contains two carboxyl groups one of which is cleaved off during the reaction. All known NMNATs may use NaMN to form a dinucleotide and the subsequent reaction with ATP then yields nicotinic acid adenine dinucleotide, NAAD. This intermediate is the substrate of NAD synthase, an enzyme 

The phosphorylation of NAD at the 2 position yields NADP and is catalysed by NAD kinase. Plant isoforms of this enzymes have been demonstrated to be r ulated by calmodulin. However, the recombinant hiunan enzyme was not directly affeaed by calcium or calmodulin. Still, this enzyme is presumably tightly regulated considering the essential roles of NADPH for reductive syntheses and especially for oxidative defence systems. 

However, it should be borne in mind that other enzymes using NAD in r ulatory reactions, including PARPs and ADP-ribosyl cyclase, are also inhibited by nicotinamide. In fact, nicotinamide has been used as PARP inhibitor in a variety of experimental settings. Perhaps, some of these studies need to be reevaluated with regard to effects of nicotinamide on other important r ulatory mechanisms such as NAD-dependent protein deacetylarion. 

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Several characterized NRPSs utilize alternative methods for chain termination. In some synthetases, the TE domain of the final module is replaced by an NAD(P)H-dependent reductase domain. Reduction of a peptidyl-S-PCP substrate through a two-electron reaction leads to the formation of a transient aldehyde, which is subsequently converted into a cyclic imine or hemiaminal through intramolecular cyclization. This two-electron reaction is utilized in the biosynthesis of nostocyclopeptides, the saframycins, ° and anthramycin. Alternatively, a four-electron reduction to the primary alcohol is observed in the biosynthesis of mycobacterial peptidolipids, linear gramicidin," " the myxalamides, lyngbyatoxin, " and myxochelin A 75,76 alternative four-electron reduction pathway involving aldehyde formation, transamination, and reduction to a primary amine occurs in the biosynthesis of myxochelin B. ... 

The oxidation state of thiazolines and oxazolines can be adjusted by additional tailoring enzymes. For instance, oxidation domains (Ox) composed of approximately 250 amino acids utilize the cofactor FMN (flavin mononucleotide) to form aromatic oxazoles and thiazoles from oxazolines and thiazolines, respectively. Such domains are likely utilized in the biosynthesis of the disorazoles, " diazonimides, bleomycin, and epothiolone. The typical domain organization for a synthetase containing an oxidation domain is Cy-A-PCP-Ox however, in myxothiazol biosynthesis one oxidation domain is incorporated into an A domain. Alternatively, NRPSs can utilize NAD(P)H reductase domains to convert thiazolines and oxazolines into thiazolidines and oxazolidines, respectively. For instance, PchC is a reductase domain from the pyochelin biosynthetic pathway that acts in trans to reduce a thiazolyinyl-Y-PCP-bound intermediate to the corresponding thiazolidynyl-Y-PCP. ... 

The reducing equivalents temporarily stored in NAD(P)H are utilized in a number of ways, all of which lead to biosynthesis of essential molecules and/or oxidative degradation of metabolites. Examples range from the simple reduction of a substrate for biosynthetic purposes (e.g. the steroid reductase mediated hydrogenation of the isolated double bond of desmosterol to give cholesterol. Table 1) (71MI11000) to complex electron transport chains that are switched on by the transfer of the electrons of NAD(P)H to the next electron carrier of the chain. These multienzyme systems are used for a number of purposes (see below). 

Figure 31 Bacillaene biosynthesis, (a) Structure of dihydrobacillaene and bacillaene. PksJ oxidizes the C14 -C15 bond after dihydrobacillaene has been synthesized. Also, note the a-hydroxyacyl N-cap. This particular N-capping has been reported very rarely. (b)a- and /3-Ketoreduction of a-KICto a-HIC. The KR domain of the first PKS module in PksJ is capable of reducing both the a-KIC amide and the /3-ketone in an NAD(P)H-dependent fashion. The order in which these two reductions occur is unknown. Ultimately, keto-reduction is followed by dehydration and enoyl reduction, (c) Theoretical structure of PPant ejection ions used to analyze PksJ ketoreduction. Right PPant ejection ion resulting from IRMPD of Acac-S-PksJ(T3-T4) incubated with PksJ. Mass shift of +2.017 Da corresponds with reduction of the /3-ketone. Left PPant ejection ion resulting from IRMPD of a-KIC-GABA-S-PksJ(T3-T4) incubated with PksJ. Shift of +2.015 Da is observed in PPant ejection ions.

The biosynthesis of NAD(P) in E. coli is outlined in Fig. 2 [3]. Oxidation of aspartic acid to the imine 14 followed by condensation with dihydroxyacetone... 

There are other mechanisms to release NRP intermediates from NRPSs, although TE domains are mostly utilized. For example, cyclosporine A is possibly released and macrocyclized by one unusual N-terminal C domain in its synthetase. Besides cyclosporine A, thaxomin A may also employ the same strategy in its biosynthesis. The another uncommon method to release NRP intermediates is to reduce the final carboxy group with the consumption of NAD(P)H by reduction domain, exemplified by nostocyclopeptide biosynthetic system. Nonetheless, TE-catalyzed macrocyclization is the favorable mechanism for product proteolytic... 

It was originally postulated that the methyl groups at C-4 were removed as COj -a suggestion that has proved to be correct. These groups are hydroxylated by a mixed-function oxidase which is NAD(P)H and Oj dependent. First, the 4a-methyl is attacked, yielding the 4 -hydroxymethyl-4 -methyl sterol. This reaction is catalyzed by a methyl sterol oxidase which has been solubilized and partially purified in Gaylor s laboratory [108]. The same enzyme preparation will, with reduced pyridine nucleotide and dioxygen, oxidize the C-30 carbon to a carboxylic acid. The 4a-methyl-4/8-hydroxymethyl-5a-cholestan-3j8-ol is not a substrate for sterol biosynthesis while its epimer is [5]. The detailed mechanisms for the enzymatic removal of C-30 and C-31 are not fully understood. The initial reaction yields a 4a-hydroxy-methyl sterol by inference however, neither the isolation nor the enzymatic formation of a 4a-hydroxymethyl sterol has been demonstrated in animal tissues. This may well result from the fact that the hydroxylation reaction is the slow step in the demethylation process [5]. 

In discussions on the mechanisms of the enzymes involved in each pathway, there will be a particular focus on three superfamilies enzymes that share the thiolase fold and catalyze carbon—carbon bond formation and cleavage reactions catalyzed by NAD(P)-dependent enzymes in the fatty acid biosynthesis pathway involve proteins that are members of the short-chain dehydrogenase reductase (SDR) superfamily and finally there are mechanistic parallels between the hydration and dehydration reactions in each pathway with a particular focus on the crotonase superfamily. 

Biosynthesis 0-Hydroxyethy )-TPP formed from pyruvic acid condenses with 2-oxobutanoic acid to give 2-acetyl-2-hydroxybutanoic acid. The latter is rearranged and reduced by acetolactate mutase (EC 5.4.99.3.) and NAD(P)H-dependent reductase to 2,3-di-hydroxy-3-methylpentanoic acid. Dehydration then furnishes 2-oxo-3-methylpentanoic which is finally transaminated to He. 

Biosynthesis Leu is formed from pyruvic acid - 2-acetolactic acid [acetolactate synthase (EC 4.1.3.18.)+(l-hydroxyethyl)-TPP] - 2,3-dihydroxy-isovaleric acid [reductase+NAD(P)H] - 2-oxoisova-leric acid [dihydroxyacid dehydratase] - 2-isopropyl-malate [2-isopropylmalate synthase + acetyl-CoA (EC 4.1.3.12)] -> 3-isopropylmalate [isopropylmalate dehydratase (EC 4.2.1.33) -HjO+HiO] 2-oxo-isocaproate [3-isopropylmalate dehydrogenase (EC 1.1.1.85) + NAD ] L. [leucine aminotransferase (EC... 

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