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

Jun Yanagidani, Takashi Tamura, Kenji Inagaki, Kenji Soda, Hidehiko Tanaka

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Abstract

A strain of Bacillus sphaericus isolated from a local soil sample has been found to use β,β-dimethyl-DL-cysteine (DL-penicillamine) as the sole nitrogen source. Crude cell extract of the bacterium showed potent penicillamine-consuming activity only in the presence of NAD, which, however, was not used as an electron acceptor. Characterization of reaction products revealed 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 product, ADP-penicillamine, caused potent product inhibition on the purified enzyme, and adenylate deaminase was found to be effective in converting the inhibitory product into inosine-diphosphate-penicillamine and thereby maintained the catalysis for several hours. The novel enzyme, termed as NAD:penicillamine ADP transferase, showed a single band on SDS-polyacrylamide gel electrophoresis with a mass of approximately 42 kDa. The native enzyme was monomeric. 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-cysteine, L-homocysteine, L-cystine, L-leucine, and L-isoleucine did not serve as the substrate. Kinetic studies suggested an Ordered Bi Bi mechanism, with 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 step in penicillamine degradation in the strain of B. sphaericus.

Original languageEnglish
Pages (from-to)795-800
Number of pages6
JournalJournal of Biological Chemistry
Volume274
Issue number2
DOIs
Publication statusPublished - Jan 8 1999

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Penicillamine
Bacilli
NAD
Bacillus
Purification
Adenosine Diphosphate
Enzymes
Inosine Diphosphate
Cysteine
AMP Deaminase
Adenosine Diphosphate Ribose
Substrates
Cystine
Isoleucine
Valine
Homocysteine
Adenosine Monophosphate
Substrate Specificity
NAD - penicillamine ADP transferase
phosphoramide

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Purification and characterization of NAD: penicillamine ADP transferase from Bacillus sphaericus: A novel NAD-dependent enzyme catalyzing phosphoramide bond formation",
abstract = "A strain of Bacillus sphaericus isolated from a local soil sample has been found to use β,β-dimethyl-DL-cysteine (DL-penicillamine) as the sole nitrogen source. Crude cell extract of the bacterium showed potent penicillamine-consuming activity only in the presence of NAD, which, however, was not used as an electron acceptor. Characterization of reaction products revealed 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 product, ADP-penicillamine, caused potent product inhibition on the purified enzyme, and adenylate deaminase was found to be effective in converting the inhibitory product into inosine-diphosphate-penicillamine and thereby maintained the catalysis for several hours. The novel enzyme, termed as NAD:penicillamine ADP transferase, showed a single band on SDS-polyacrylamide gel electrophoresis with a mass of approximately 42 kDa. The native enzyme was monomeric. 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-cysteine, L-homocysteine, L-cystine, L-leucine, and L-isoleucine did not serve as the substrate. Kinetic studies suggested an Ordered Bi Bi mechanism, with 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 step in penicillamine degradation in the strain of B. sphaericus.",
author = "Jun Yanagidani and Takashi Tamura and Kenji Inagaki and Kenji Soda and Hidehiko Tanaka",
year = "1999",
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T1 - Purification and characterization of NAD

T2 - penicillamine ADP transferase from Bacillus sphaericus: A novel NAD-dependent enzyme catalyzing phosphoramide bond formation

AU - Yanagidani, Jun

AU - Tamura, Takashi

AU - Inagaki, Kenji

AU - Soda, Kenji

AU - Tanaka, Hidehiko

PY - 1999/1/8

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N2 - A strain of Bacillus sphaericus isolated from a local soil sample has been found to use β,β-dimethyl-DL-cysteine (DL-penicillamine) as the sole nitrogen source. Crude cell extract of the bacterium showed potent penicillamine-consuming activity only in the presence of NAD, which, however, was not used as an electron acceptor. Characterization of reaction products revealed 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 product, ADP-penicillamine, caused potent product inhibition on the purified enzyme, and adenylate deaminase was found to be effective in converting the inhibitory product into inosine-diphosphate-penicillamine and thereby maintained the catalysis for several hours. The novel enzyme, termed as NAD:penicillamine ADP transferase, showed a single band on SDS-polyacrylamide gel electrophoresis with a mass of approximately 42 kDa. The native enzyme was monomeric. 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-cysteine, L-homocysteine, L-cystine, L-leucine, and L-isoleucine did not serve as the substrate. Kinetic studies suggested an Ordered Bi Bi mechanism, with 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 step in penicillamine degradation in the strain of B. sphaericus.

AB - A strain of Bacillus sphaericus isolated from a local soil sample has been found to use β,β-dimethyl-DL-cysteine (DL-penicillamine) as the sole nitrogen source. Crude cell extract of the bacterium showed potent penicillamine-consuming activity only in the presence of NAD, which, however, was not used as an electron acceptor. Characterization of reaction products revealed 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 product, ADP-penicillamine, caused potent product inhibition on the purified enzyme, and adenylate deaminase was found to be effective in converting the inhibitory product into inosine-diphosphate-penicillamine and thereby maintained the catalysis for several hours. The novel enzyme, termed as NAD:penicillamine ADP transferase, showed a single band on SDS-polyacrylamide gel electrophoresis with a mass of approximately 42 kDa. The native enzyme was monomeric. 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-cysteine, L-homocysteine, L-cystine, L-leucine, and L-isoleucine did not serve as the substrate. Kinetic studies suggested an Ordered Bi Bi mechanism, with 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 step in penicillamine degradation in the strain of B. sphaericus.

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