4904
J. Am. Chem. Soc. 1999, 121, 4904-4905
Formation of Nicotinamide from Nicotinuric Acid by
Peptidylglycine r-Amidating Monooxygenase
(PAM): A Possible Alternative Route from Nicotinic
Acid (Niacin) to NADP in Mammals
David J. Merkler,*,† Uta Glufke,‡
Kimberly J. Ritenour-Rodgers,† Laura E. Baumgart,†
Jodi L. DeBlassio,† Kathleen A. Merkler,† and
John C. Vederas*,‡
Department of Chemistry and Biochemistry
Duquesne UniVersity, Pittsburgh, PennsylVania 15282
Department of Chemistry, UniVersity of Alberta
Edmonton, Alberta, Canada T6G 2G2
ReceiVed February 18, 1999
Peptidylglycine R-amidating monooxygenase (PAM, EC
1.14.17.3) is a copper and zinc-dependent enzyme that generates
neuropeptides having a C-terminal amide functionality by oxida-
tive removal of the two carbons from a glycine-extended
precursor.1,2 The process probably occurs in all animals3 and
requires two steps (Figure 1). The first involves ascorbate and
copper-dependent aerobic hydroxylation of the glycine R-carbon
with retention of configuration by peptidylglycine R-hydroxylating
monooxygenase (PHM). The second step, catalyzed by peptidyl-
amidoglycolate lyase (PAL), is zinc-dependent and resembles a
retro-aldol reaction to form the primary amide and glyoxylate.
In mammals, RNA splicing and posttranslational modifications
lead to bifunctional PAM as well as monofunctional PHM and
PAL proteins.2 An X-ray analysis of the core of PHM with a
bound substrate provides an intriguing picture of its catalytic
machinery, which has two copper atoms about 11 Å apart in the
active site.4,5 Recently, it has become clear that PAM substrate
specificity for the N-acyl moiety on the glycine extends beyond
peptides to fatty acids and other lipids.6,7 For example, the natural
sleep-inducing substance, oleamide,8 may be generated in mam-
mals by PAM cleavage of N-oleylglycine.6a Since a host of
carboxylic acids can be conjugated to glycine in vivo by acyl-
CoA:glycine N-acyltransferase (ACGNAT, EC 2.3.1.13)9 and
nicotinic acid (niacin, vitamin B3) (1) is known to be rapidly
Figure 1. Function of amidating enzymes and structures of nicotinic
acid derivatives.
converted in the liver to nicotinuric acid (2),10 it was of interest
to determine whether 2 could be transformed to nicotinamide (3)
by PAM. In the present study, we describe this conversion as
well as preliminary labeling studies with rats to evaluate the
importance of this pathway in mammals for the formation of
NAD(P).11
Recombinant type A rat medullary carcinoma PAM was
purified6b,12 and shown to cleave 2 to 3 and glyoxylate. Production
of the glyoxylate was determined spectrophotometrically after
formation of its 1,5-diphenylformazan derivative,6b,13 and nico-
tinamide formation was shown by HPLC analysis.13 Oxygen
consumption was measured with an electrode6b to provide kinetic
information for the initial oxidative step (KM,
) 1.9 ( 0.14
app
mM; V/Kapp ) 4.3 ( 0.24 × 103 M-1 s-1). Under similar
conditions, the V/Kapp values for other PAM substrates are 4.6 (
0.087 × 104 M-1 s-1 for D-Tyr-Val-Gly, 1.7 ( 0.055 × 105 M-1
s-1 for N-lauroylglycine, and 6.2 ( 0.12 × 103 M-1 s-1 for
N-benzoylglycine. To examine this process by NMR spectrometry,
[glycyl-1,2-13C2]-nicotinuric acid 2a was synthesized by coupling
the corresponding labeled glycine (99 atom % 13C) to nicotinic
acid azide (4).15 Reaction of PAM and 2a in deuterated buffer
(pD 5.6) with copper and ascorbate under aerobic conditions was
followed by HMQC NMR analysis.16 These spectra show that
the only detectable labeled species are the starting material 2a
and the hydrate of [1,2-13C2]-glyoxylate. However, the same reac-
tion in the presence of the known PAL inhibitor, 2,4-dioxo-5-
acetamido-6-phenylhexanoic acid (N-Ac-Phe-pyruvate) (5),17
† Duquesne University.
‡ University of Alberta.
(1) Bradbury, A. F.; Finnie, M. D. A.; Smyth, D. G. Nature (London) 1982,
298, 686-688.
(2) For reviews, see: (a) Kulathila, R.; Merkler, K. A.; Merkler, D. J. Nat.
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E. Annu. ReV. Neurosci. 1992, 15, 57-85.
(3) (a) Zabriskie, T. M.; Klinge, M.; Szymanski, C. M.; Cheng, H.; Vederas,
J. C. Arch. Insect Biochem. Physiol. 1994, 26, 27-48. (b) Kolhekar, A. S.;
Roberts, M. S.; Jiang, N.; Johnson, R. C.; Mains, R. E.; Eipper, B. A.; Taghert,
P. H. J. Neurosci. 1997, 17, 1363-1376. (c) Grimmelikhuijzen, C. J. P.;
Leviev, I.; Carstensen, K. Int. ReV. Cytol. 1996, 167, 37-89.
(4) Prigge, S. T.; Kolhekar, A. S.; Eipper, B. A.; Mains, R. E.; Amzel, L.
M. Science 1998, 278, 1300-1305.
(9) (a) Bartlett, K.; Gompertz, D. Biochem. Med. 1974, 10, 15-23. (b)
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T. Benet, L. Z. Drug Metab. Dispos. 1996, 24, 773-779.
(5) PHM has homology to dopamine â-monooxygenase: Francisco, W.
A.; Merkler, D. J.; Blackburn, N. J.; Klinman, J. P. Biochemistry 1998, 37,
8244-8252.
(6) (a) Merkler, D. J.; Merkler, K. A.; Stern, W.; Fleming, F. F. Arch.
Biochem. Biophys. 1996, 330, 430-434. (b) Wilcox, B. J.; Ritenour-Rodgers,
J.; Asser, A. S.; Baumgart, L. E.; Baumgart, M. A.; Boger, D. L.; DeBlassio,
J. L.; deLong, M. A.; Glufke, U.; Henz, M. E.; King, L., III; Kulathila, R.;
Merkler, K. A.; Patterson, J. E.; Robleski, J. J.; Vederas, J. C.; Merkler, D. J.
Biochemistry 1999, 38, 3235-3245.
(11) For reviews of NAD(P) biosynthesis, see: (a) Ijichi, H.; Ichiyama,
A.; Hayaishi, O. J. Biol. Chem. 1966, 241, 3701-3707. (b) Moat, A. G.;
Foster, J. W. Pyridine Nucleotide Coenzymes, Part B. In Coenzymes and
Cofactors; Dolphin, D., Poulson, R., Avramovic, O., Eds.; Wiley: New York,
1987; Vol II, pp 1-24. (c) DiPalma, J. R. In Vitamins; Friedrich, W., Ed.;
Walter de Gruyter: Berlin, 1988; pp 473-542.
(7) Specificity for the glycine skeleton is high but some D-amino acids
can be substituted: Andrews, M. D.; O’Callaghan, K. A.; Vederas, J. C.
Tetrahedron 1997, 53, 8295-8306.
(12) Miller, D. A.; Sayad, K. U.; Kulathila, R.; Beaudry, G. A.; Merkler,
D. J.; Bertelsen, A. H. Arch. Biochem. Biophys. 1992, 298, 380-388.
(13) Katopodis, A. G.; May, S. W. Biochemistry 1990, 29, 4541-4548.
(14) Nicotinuric acid, N-nicotinoyl-R-hydroxyglycine, and nicotinamide
were resolved at 50 °C on a Keystone Scientific ODS Hypersil column (100
× 4.6 mm). Isocratic elution was achieved using a mobile phase of 200 mM
sodium acetate pH 6.6, and analytes were detected by UV absorbance at 246
nm.
(8) (a) Lerner, R. A.; Siuzdak, G.; Prospero-Garcia, O.; Henriksen, S. J.;
Boger, D. L.; Cravatt, B. F. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 9505-
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Henriksen, S. J.; Boger, D. L.; Lerner, R. A. Science 1995, 268, 1506-1509.
(c) Boger, D. L.; Henriksen, S. J.; Cravatt, B. F. Curr. Pharm. Des. 1998, 4,
303-314.
(15) Rohrlich, M. Arch. Pharm. (Weinheim, Ger.) 1951, 284, 6-7.
10.1021/ja990517e CCC: $18.00 © 1999 American Chemical Society
Published on Web 05/11/1999