Interaction of Rv1625c, a mycobacterial class IIIa adenylyl cyclase, with a mammalian congener.
The adenylyl cyclase Rv1625c from Mycobacterium tuberculosis codes for a protein with six transmembrane spans and a catalytic domain, i.e. it corresponds to one half of the pseudoheterodimeric mammalian adenylyl cyclases (ACs). Rv1625c is active as a homodimer. We investigated the role of the Rv1625c membrane domain and demonstrate that it efficiently dimerizes the protein resulting in a 7.5-fold drop in K(m) for ATP. Next, we generated a duplicated Rv1625c AC dimer by a head-to-tail concatenation. This produced an AC with a domain order exactly as the mammalian pseudoheterodimers. It displayed positive cooperativity and a 60% increase of v(max) compared with the Rv1625c monomer. Further, we probed the compatibility of mycobacterial and mammalian membrane domains. The second membrane anchor in the Rv1625c concatamer was replaced with membrane domain I or II of rabbit type V AC. The mycobacterial and either mammalian membrane domains are compatible with each other and both recombinant proteins are active. A M. tuberculosis Rv1625c knockout strain was assayed in a mouse infection model. In vitro growth characteristics and in vivo organ infection and mortality were unaltered in the knockout strain indicating that AC Rv1625c alone is not a virulence factor.
Guo
Ying Lan
YL
Abt. Pharmazeutische Biochemie, Pharmazeutisches Institut, Universität Tübingen, Morgenstelle 8, D-72076 Tübingen, Germany.
Kurz
Ursula
U
Schultz
Anita
A
Linder
Jürgen U
JU
Dittrich
Dorothea
D
Keller
Christine
C
Ehlers
Stefan
S
Sander
Peter
P
Schultz
Joachim E
JE
D000262:Adenylyl Cyclases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D020134:Catalytic Domain; D019281:Dimerization; D004195:Disease Models, Animal; D005260:Female; D006801:Humans; D007700:Kinetics; D051379:Mice; D008810:Mice, Inbred C57BL; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014397:Tuberculosis, Pulmonary / Q000382:microbiology / Q000503:physiopathology*; D014774:Virulence
D011993:Recombinant Fusion Proteins; D000262:Adenylyl Cyclases