Abstract
Mycobacterium tuberculosis contains 15 class III adenylyl cyclase genes. The gene Rv1264 is predicted to be composed of two distinct protein modules. The C terminus seems to code for a catalytic domain belonging to a subfamily of adenylyl cyclase isozymes mostly found in Gram-positive bacteria. The expressed protein was shown to function as a homodimeric adenylyl cyclase (1 micromol of cAMP x mg(-1) x min(-1)). In analogy to the structure of the mammalian adenylyl cyclase catalyst, six amino acids were targeted by point mutations and found to be essential for catalysis. The N-terminal region represents a novel protein domain, the occurrence of which is restricted to several adenylyl cyclases present in Gram-positive bacteria. The purified full-length enzyme was 300-fold less active than the catalytic domain alone. Thus, the N-terminal domain appeared to be autoinhibitory. The N-terminal domain contains three prominent polar amino acid residues (Asp(107), Arg(132), and Arg(191)) that are invariant in all seven sequences of this domain currently available. Mutation of Asp(107) to Ala relaxed the inhibition and resulted in a 6-fold increase in activity of the Rv1264 holoenzyme, thus supporting the role of this domain as a potential novel regulator of adenylyl cyclase activity.
Highlights
CAMP serves as a second messenger in virtually all organisms; yet at least three independent classes of adenylyl cyclases (AC)[1] exist
Alignment with other bacterial class III cyclase homology domain (CHD) of this subfamily and with the mammalian-type AC Rv1625c from M. tuberculosis demonstrates the presence of all six amino acids in register in a single polypeptide chain that have been identified by x-ray data to be critical for catalysis in the mammalian pseudoheterodimeric ACs
What follows is a stretch of about 50 amino acids, which again is highly variable in length, in essentially all CHDs irrespective of their origin
Summary
Materials—Genomic DNA from M. tuberculosis was provided by Dr Boettger (University of Zurich Medical School). The following additional restriction sites were introduced by silent mutations: a StuI site at base position 411 for construc- The expression plasmid for the N-terminal domain was generated from pQE30 containing Rv1264(1–397) by digestion with NaeI and circularization of the largest fragment. This created an expression cassette of Rv1264(1–207) with an N-terminal His[6] tag and a C-terminal SSP tripeptide extension. 250 l of nickel-nitrilotriacetic acid-agarose equilibrated in 5 ml of buffer A (lysis buffer containing 250 mM NaCl, 15 mM imidazole, 5 mM MgCl2) were added, and the protein was allowed to bind for 60 min. The proteins were blotted onto a polyvinylidene difluoride membrane and visualized with an anti-RGS-His[4] antibody (3)
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