Purification and characterization of the enzymes involved in nicotinamide adenine dinucleotide degradation by Penicillium brevicompactum NRC 829

Abstract

The present study was conducted to investigate a new pathway for the degradation of nicotinamide adenine dinucleotide (NAD) by Penicillium brevicompactum NRC 829 extracts. Enzymes involved in the hydrolysis of NAD, i.e. alkaline phosphatase, aminohydrolase and glycohydrolase were determined. Alkaline phosphatase was found to catalyse the sequential hydrolysis of two phosphate moieties of NAD molecule to nicotinamide riboside plus adenosine. Adenosine was then deaminated by aminohydrolase to inosine and ammonia. While glycohydrolase catalyzed the hydrolysis of the nicotinamide-ribosidic bond of NAD+ to produce nicotinamide and ADP-ribose in equimolar amounts, enzyme purification through a 3-step purification procedure revealed the existence of two peaks of alkaline phosphatases, and one peak contained deaminase and glycohydrolase activities. NAD deaminase was purified to homogeneity as estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis with an apparent molecular mass of 91 kDa. Characterization and determination of some of NAD aminohydrolase kinetic properties were conducted due to its biological role in the regulation of cellular NAD level. The results also revealed that NAD did not exert its feedback control on nicotinamide amidase produced by P. brevicompactum.

Extracellular Hydrolysis of NAD+and its Secondary Metabolites by a new Aspergillus sp

“…Results of Table 3 show that kcat/Km value of adenosine 1.23 x 10 -5 M -1 swhich is less than kcat/Km value in case of NAD and AMP. In addition kcat/Km value of adenosine was also less than the other purine and pyrimidine nucleoside degradation in both of A. oryzae and P.brevi compactum [20][21].Therefore, it becomes more efficient, and hence more products are generated at a faster rate than in other microorganisms.…”

“…In accordance with the assumption presence of 5'-nucleotidase that cleavage MNM and AMP to nicotinamide riboside and adenosine as products chromatographically detected in each of the different reaction mixtures.This 5'-nucleotidase has acidic natured activity like A oryzae acid phosphatase [20] but it differs from Penicillium brevicompactum, Escherichia coli enzymes [21,9] that the two later organisms having optimum activity at alkaline reactions. Appearance of inosine and adenosine in chromatographic examination illustrated the partial d eamination of adenosine as the results has been found in extracts of A. niger [18], A. terreus [41]and A. oryzae [19] and Penicillium brevicompactum, [21]. From chromatographic analysis, No ability of the pure aminohydrolase to deaminate the amide group of each nicotinamide or nicotinamide riboside…”

“…Acid phosphatase is detailed to be found within the cell wall and septa of hyphae as decided by cytochemical studies by transmission electron microscopy (TEM) [17].In some filamentous fungi, acid phosphatases involved in NAD hydrolysis to nicotinamide riboside and adenosine as have been characterized in our laboratory from A. niger [18], A.orysae [19][20]and P. brevicompactum [21]. The acid phosphatase, at the these previous fungal strains split ADP to AMP furthermore free inorganic phosphates.…”

The essential schistosome tegumental ectoenzyme SmNPP5 can block NAD-induced T cell apoptosis

“…Aliquots from enzyme reaction mixtures, as well as chemical standards β-NAD (Roche), AMP and NMN(Sigma, A2252 and N3501),were spotted onto a TLC sheet and dried. Separation was achieved using a mobile phase composed of n-butanol:acetone:acetic acid (glacial):ammonia (5%):water (45:15:10:10:20) and analytes were visualized under UV at 254 nm [ 31 ].…”