The gene Rv1625c from Mycobacterium tuberculosis encodes a membrane-anchored adenylyl cyclase corresponding to exactly one-half of a mammalian adenylyl cyclase. An engineered, soluble form of Rv1625c was expressed in Escherichia coli. It formed a homodimeric cyclase with two catalytic centers. Amino acid mutations predicted to affect catalysis resulted in inactive monomers. A single catalytic center with wild-type activity could be reconstituted from mutated monomers in stringent analogy to the mammalian heterodimeric cyclase structure. The proposed existence of supramolecular adenylyl cyclase complexes was established by reconstitution from peptide-linked, mutation-inactivated homodimers resulting in pseudo-trimeric and -tetrameric complexes. The mycobacterial holoenzyme was expressed successfully in E.coli and mammalian HEK293 cells, i.e. its membrane targeting sequence was compatible with the bacterial and eukaryotic machinery for processing and membrane insertion. The membrane-anchored mycobacterial cyclase expressed in E.coli was puri®ed to homogeneity as a ®rst step toward the complete structural elucidation of this important protein. As the closest progenitor of the mammalian adenylyl cyclase family to date, the mycobacterial cyclase probably was spread by horizontal gene transfer.
Proregions of papain-like cysteine proteases are potent and often highly selective inhibitors of their parental enzymes. The molecular basis of their selectivity is poorly understood. For two closely related members of the cathepsin Llike subfamily we established strong selectivity differences. The propeptide of cathepsin S was observed to inhibit cathepsin L with a K i of 0.08 nM, yet cathepsin L propeptide inhibited cathepsin S only poorly. To identify the respective structural correlates we engineered chimeric propeptides and compared their inhibitory specificity with the wild-types. Specificity resided in the N-terminal part, strongly suggesting that the backbone of the prodomain was the underlying structure.z 2000 Federation of European Biochemical Societies.
SummaryThe 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.
Mammalian oocytes play an important role in the reproductive process (Yamada & Isaji, 2011). Recently, due to rapid development of in vitro fertilization (IVF) and cloning techniques, oocyte maturation has become an essential topic in life science. Mammalian oocyte maturation in follicles is considered a complex process, since a variety of signalling proteins and molecules participate and form a signal transduction pathway (Minami & Tsukamoto, 2006). Studying the maturation mechanisms of oocytes from the level of gene expression and protein regulation will help to optimize the in vitro culture system of oocytes, improving the in vitro maturation efficiency and embryo production efficiency. Therefore, identification and investigation of oocyte proteins will be of obvious significance to understanding oocyte maturation, fertilization and embryonic development (Minami & Tsukamoto, 2006). Equally, examination of the mammalian oocyte proteome is of great significance for understanding the molecular mechanisms of oocyte maturation. At present, mammalian oocyte/embryo proteomics has been performed on mice (Ma
cAMP generation in bacteria is often stimulated by sudden, but lasting, changes in extracellular conditions, whereas intracellular cAMP concentrations quickly settle at new levels. As bacteria lack G‐proteins, it is unknown how bacterial adenylate cyclase (AC) activities are modulated. Mycobacterium tuberculosis has 15 class III AC genes; therefore, we examined whether mycobacteria contain a factor that may regulate AC activities. We identified mycobacterial polyphosphates with a mean chain length of 72 residues as highly potent inhibitors of dimeric class IIIa, class IIIb and class IIIc ACs from M. tuberculosis and other bacteria. The identity of the inhibitor was established by phosphatase degradation, 31P‐NMR, acid or base hydrolysis, PAGE and comparisons with commercial standards, and functional substitution by several polyphosphates. The data indicate that each AC dimer occupies 8–15 phosphate residues on a polyphosphate strand. Other polyionic polymers such as polyglutamate, polylysine and hyaluronic acid do not affect cyclase activity. Notably, the structurally unrelated class I AC Cya from Escherichia coli is unaffected. Bacterial polyphosphate metabolism is generally viewed in the context of stress‐related regulatory networks. Thus, regulation of bacterial class III ACs by polyphosphates could be a component of the bacterial stress response.
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