Cytosolic NADP phosphatases I and II from Arthrobacter sp. strain KM: implication in regulation of NAD+/NADP+ balance.
NADP phosphatase (NADPase) is an enzyme that converts NADP+ into NAD+ through dephosphorylation of NADP+, and is considered to be one of the possible candidates for regulation of the NAD+/NADP+ balance in vivo. In order to obtain an intrinsic NADPase, the NADP+-degrading activity in a membrane-free cell extract of a Gram-positive bacterium, Arthrobacter sp. strain KM, was first assessed and demonstrated to be mainly achieved through the NADPase reaction, indicating NADPase is essential for degradation of NADP+ and therefore for regulation of the NAD+/NADP+ balance in cytosol. Then, the isolation of cytosolic NADPase was attempted using NADP+ as a substrate. Two NADPase isozymes, designated as NADPases I and II, were purified from the cell extract of the bacterium, and were indicated to be the sole cytosolic NADPases regulating the balance of NAD+/NADP+. NADPases I and II are homodimers of 32 and 30 kDa subunits, respectively, and most active at pH 7-8. The N-terminal amino acid sequences of the two enzymes are similar to each other. Among the biological substrates tested, both enzymes showed the highest activity toward NADP+ and NADPH. AMP, ADP, and pyridoxal 5'-phosphate were also dephosphorylated, but to lower extents. Comparison of the features of NADPases I and II with those of other acid phosphatases possessing NADPase activity suggested that NADPases I and II are novel enzymes participating in regulation of the NAD+/NADP+ balance in the cytosol.
Kawai
Shigeyuki
S
Department of Basic and Applied Molecular Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan.
Mori
Shigetarou
S
Mukai
Takako
T
Murata
Kousaku
K
D000135:Acid Phosphatase / Q000502:physiology; D000244:Adenosine Diphosphate / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001173:Arthrobacter / Q000201:enzymology*; D003593:Cytoplasm / Q000201:enzymology*; D019281:Dimerization; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D009708:Nucleotidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D021122:Protein Subunits / Q000737:chemistry; D011732:Pyridoxal Phosphate / Q000378:metabolism; D013379:Substrate Specificity
D007527:Isoenzymes; D021122:Protein Subunits; D009243:NAD; D000249:Adenosine Monophosphate; D009249:NADP; D011732:Pyridoxal Phosphate; D000244:Adenosine Diphosphate; C045660:NADP phosphatase; D009708:Nucleotidases; D000135:Acid Phosphatase