Purification and characterization of NAD : penicillamine ADP transferase from Bacillus sphaericus - A novel NAD-dependent enzyme catalyzing phosphoramide bond formation

A strain of Bacillus sphaericus isolated from a local soil sample has been found to use beta,beta-dimethyl-DL-cysteine (DL-penicillamine) as the sole nitrogen source, Crude cell extract of the bacterium showed potent penicill amine-consuming activity only in the presence of NAD, which, however, was n ot used as an electron ac ceptor, Characterization of reaction products rev ealed that penicillamine was derivatized to a phosphoramide adduct with the ADP moiety of NAD, whereas the nicotinamide-ribose group was released and hydrolyzed spontaneously to ribose and nicotinamide, The phosphoramide prod uct, ADP-penicillamine, caused potent product inhibition on the purified en zyme, and adenylate deaminase was found to be effective in converting the i nhibitory product into inosine-diphosphate-penicillamine and thereby mainta ined the catalysis for several hours, The novel enzyme, termed as NAD:penic illamine ADP transferase, showed a single band on SDS-polyacrylamide gel el ectrophoresis with a mass of approximately 42 kDa, The native enzyme was mo nomeric. The enzyme showed high substrate specificity to NAD (K-m = 13.0 mM ) and L-penicillamine (K-m = 6.5 mM); other nucleotides such as NADP, NAD(P )H, AMP, ADP, and ADP-ribose did not substitute for NAD, and L-valine, L-cy steine, L-homocysteine, L-cystine, L-leucine, and L-isoleucine did not serv e as the substrate. Kinetic studies suggested an Ordered Bi Bi mechanism, w ith NAD as the first substrate to bind and ADP-L-penicillamine as the last product released, The novel NAD-dependent enzyme may catalyze the first ste p in penicillamine degradation in the strain of B. sphaericus.