718 Medicinal Chemistry, 2011, 7, 718-726
1573-4064/11 $58.00+.00 © 2011 Bentham Science Publishers
Cloning, Expression and Characterization of Mouse (Mus musculus)
Nicotinamide 5'-Mononucleotide Adenylyltransferase-2
David W. Raches
1
, Suhong Xiao
2
, Praveen Kusumanchi
3
, Joel Yalowitz
2
, Paresh Sanghani
1
, Eric C. Long
4
,
Aok C. Antony
2,3
and Hiremagalur N. Jayaram
1,3,
*
1
Department of Biochemistry and Molecular Biology, and
2
Department of Medicine, Indiana University School of Medi-
cine, Indianapolis, USA;
3
Richard L. Roudebush Veterans Affairs Medical Center, Indianapolis, USA;
4
Department of
Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis (IUPUI), Indianapolis, USA
Abstract: Nicotinamide adenine dinucleotide (NAD
+
) is synthesized by the action of nicotinamide mononucleotide
adenylyltransferase (NMNAT) from NMN and ATP. The mouse homolog of NMNAT-2 (mmNMNAT-2) was cloned,
expressed, and subsequently identified using MALDI-TOF in conjunction with the ProFound database. Circular dichroism
analyses of recombinant mmNMNAT-2 showed helical and sheet secondary structures, consistent with the known
structure of the human isoform. Competition experiments using mouse pancreatic tissue lysates with recombinant
mmNMNAT-2 demonstrated that the activity of the expressed protein was similar to the human isoform. Immunohisto-
chemistry of mouse embryonic tissues with hNMNAT-2 also showed a tissue- and cellular-specific expression of this iso-
form. Therefore, our studies demonstrate for the first time the clear biological evidence for the existence of a mouse iso-
form of hNMNAT-2. These studies may help in future investigations aimed at understanding the regulation of this gene
and its pathway, and in turn, will spur the development of novel therapies for diseases such as cancer and diabetes since
mice are the most frequently used experimental system for in vivo studies.
Keywords: Circular dichroism, MALDI-TOF, mouse NMNAT-2, tissue expression, NAD, tiazofurin.
INTRODUCTION
Many of the enzymatic reactions that mediate cellular
processes require high-energy electron carrier nucleotides
such as nicotinamide adenine dinucleotide (NAD
+
). Thus,
the biosynthetic pathways leading to these essential carriers
provide potential loci for anti-cancer drug development and
interdiction. NAD
+
required for cellular viability is synthe-
sized by the enzymatic action of nicotinamide mononucleo-
tide adenylyltransferase (NMNAT) from nicotinamide
mononucleotide (NMN) and adenosine triphosphate (ATP)
[1]. Biosynthesis of NAD
+
occurs either by: (1) a double
displacement reaction leading to pyrophosphate (PP
i
) release
and transfer of an adenylyl group to NMN; or by (2) a nu-
cleophilic attack of the 5'-phosphate of the NMN on the -
phosphate of ATP releasing pyrophosphate and the product
dinucleotide (Fig. 1a). Two conserved pathways for NAD
+
synthesis i.e., de novo and salvage pathways, respectively,
depend upon NMNAT to catalyze the final, rate-limiting
reaction of NMN or nicotinic acid mononucleotide with ATP
to form NAD
+
or nicotinic acid adenine dinucleotide and PP
i
(Fig. 1b) underscoring the fundamental importance of this
enzyme [2].
The reactive portion of NAD
+
is the nicotinamide ring, a
pyridine derivative synthesized from vitamin B6 (niacin).
NAD
+
is utilized as a substrate for many enzymes such as
*Address correspondence to this author at the Richard L. Roudebush Veter-
ans Affairs Medical Center-151, 1481 West Tenth street, Indianapolis, Indi-
ana, 46202, USA; Tel: 317-988-2479; Fax: 317-988-3180;
E-mail: hjayaram@iupui.edu
O
N O
OH HO
P
O
O
-
-
O
O
NH
2
O
N O
OH HO
P
O
O
-
O
+
P O
O
-
O
P
O
-
-
O
O
NMN
ATP
N
N
N
NH
2
O
O
OH HO
P
O
O
-
O P O
O
-
O
N
O
NH
2 +
O
N
HO
HO
N N
N
NH
2
NAD
+
PP
i
O
-
P O
O
-
O
P
O
-
-
O
O
+
Fig. (1a). Schematic illustration of NMNAT’s enzymatic reaction
to form NAD
+
and PP
i
from NMN and ATP.
L-Tryptophan
Quinolinic acid
Nicotinic acid
mononucleotide
Nicotinic acid adenine
dinucleotide (NaAD)
Nicotinic acid
(Na)
Nicotinamide
(Nam)
Nicotinamide
mononucleotide
(NMN)
Nicotinamide riboside
(NR)
NRK
NaPRTase
NamPRTase
NMNAT
NAD
+
NAD Synthase
QAPRTase NMNAT
de novo Pathway
Salvage Pathways
Fig. (1b). Illustration of the biosynthetic pathways for NAD
synthesis. QAPRTase: quinolinic acid phosphoribosyltransferases;
NAD synthase: nicotinamide adenine dinucleotide synthase; NaPR-
Tase: nicotinic acid phosphoribosyltransferase; NamPRTase:
nicotinamide phosphoribosyltransferase; NRK: nicotinamide ribose
kinase.