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.