A Structural Basis for the Role of Nucleotide Specifying
Residues in Regulating the Oligomerization of the
Rv1625c Adenylyl Cyclase from M. tuberculosis
Amit D. Ketkar
1,2
, Avinash R. Shenoy
1
, Udupi A. Ramagopal
3
Sandhya S. Visweswariah
1
*
and Kaza Suguna
2
*
1
Department of Molecular
Reproduction, Development and
Genetics, Indian Institute of
Science, Bangalore, 560012
India
2
Molecular Biophysics Unit
Indian Institute of Science
Bangalore 560012, India
3
Department of Biochemistry
Albert Einstein College of
Medicine, Morris Park Avenue
Bronx, NY 10461, USA
The Rv1625c Class III adenylyl cyclase from Mycobacterium tuberculosis is a
homodimeric enzyme with two catalytic centers at the dimer interface, and
shows sequence similarity with the mammalian adenylyl and guanylyl
cyclases. Mutation of the substrate-specifying residues in the catalytic
domain of Rv1625c, either independently or together, to those present in
guanylyl cyclases not only failed to confer guanylyl cyclase activity to
the protein, but also severely abrogated the adenylyl cyclase activity of
the enzyme. Biochemical analysis revealed alterations in the behavior of the
mutants on ion-exchange chromatography, indicating differences in the
surface-exposed charge upon mutation of substrate-specifying residues.
The mutant proteins showed alterations in oligomeric status as compared
to the wild-type enzyme, and differing abilities to heterodimerize with the
wild-type protein. The crystal structure of a mutant has been solved to
a resolution of 2.7 A
˚
. On the basis of the structure, and additional
biochemical studies, we provide possible reasons for the altered properties
of the mutant proteins, as well as highlight unique structural features of the
Rv1625c adenylyl cyclase.
q 2005 Elsevier Ltd. All rights reserved.
Keywords: nucleotide cyclase; Mycobacterium tuberculosis; cAMP; crystal
structure
*Corresponding author
Introduction
Cellular signal transduction involves a number of
signaling pathways, several of which cross-talk
with each other giving rise to a large signaling
network. Class III nucleotide cyclases are enzymes
involved in synthesizing 3
0
,5
0
-cyclic nucleotides
which act as important second messengers in a
variety of signal transduction processes, for
example, those involving the G-protein coupled
receptors, and the receptor and soluble guanylyl
cyclases in eukaryotes. Since the maintenance of
intracellular cyclic nucleotide levels is critical,
several studies have focused on the nucleotide
cyclases, enzymes involved in the synthesis and
degradation of cAMP and cGMP, with particular
emphasis on understanding their catalytic mechan-
isms and regulation.
The wealth of biochemical and structural infor-
mation available on eukaryotic Class III adenylyl
cyclases has shed light on the nature of the active
site of these enzymes. The mammalian adenylyl
cyclase is a head-to-tail dimer of two similar Class
III domains (C1 and C2), and has a single active site
at the dimer interface, which is formed by optimal
juxtaposing of specific residues from the
C1 and C2 domains.
1,2
In the absence of structural
information on the guanylyl cyclases, homology
modeling suggested that they too could have a
similar overall structure and that a few critical
residues could be responsible for the preferential
binding of GTP in these enzymes.
3
Such studies
indicated the role of conserved Lys, Asp and Gln
residues in mammalian adenylyl cyclases, which
are replaced, respectively, by Glu, Cys and Arg in
the guanylyl cyclases. Site-directed mutagenesis
involving swapping of two or more of these
residues in adenylyl and guanylyl cyclases from
0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
Abbreviations used: AC, adenylyl cyclase; GC,
guanylyl cyclase; GST, glutathione-S-transferase; PDB,
Protein Data Bank.
E-mail addresses of the corresponding authors:
sandhya@mrdg.iisc.ernet.in; suguna@mbu.iisc.ernet.in
doi:10.1016/j.jmb.2005.12.017 J. Mol. Biol. (2006) 356, 904–916