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.

Keywords: Alkaline phosphatases; Deaminase; Glycohydrolase; NAD degradation; Penicillium brevicompactum NRC 829.

Fig. 1

Optimal pH of NAD degrading enzymes. The reaction mixture contained the following: substrate, NAD, 5 µmol; buffers of various pHs: 80 µmol (pH 3–6) citrate-buffer, (pH 6–9) Tris–acetate, (pH 9–10) carbonate–bicarbonate buffer; temp., 50 °C; protein extracts, 2.5 mg

Fig. 2

TLC analysis of products from NAD degradation activity of P. brevicompactum aminohydrolase. Right lane was the sample of reaction mixture containing 5 µM NAD being treated at 50 °C for 2 h by aminohydrolase at an initial concentration of 20 µg. Concentrations of chemical standards were 2 mM. Abbreviations of authentic: NAD, nicotinamide adenine dinucleotide; AMP, adenosine 5′-monophosphate; ADP, adenosine 5′-diphosphate; Ado, adenosine; Ade, adenine; Ino, inosine; Nm, nicotinamide; NR, nicotinamide riboside

Fig. 3

Electrophoretic analysis of Penicillium brevicompactum NRC 829 NAD aminohydrolase. From left to right: lane 1 molecular mass markers, lane 2 fractional precipitation by chilled acetone, lane 3 partial purified NAD aminohydrolase on DEAE-Sephadex A-25, lane 4 purified NAD aminohydrolase on Sephadex G-100

Fig. 4

pH dependence of the purified deaminase activity

Fig. 5

pH stability of the NAD aminohydrolase. The enzyme was stored in buffers of various pHs (100 mM) at 50 °C for 30 min, and the residual activities were measured

Fig. 6

Effect of temperature on purified deaminase