An adenylyl cyclase pseudogene in Mycobacterium tuberculosis has a functional ortholog in Mycobacterium avium

Shenoy, Avinash R.; Srinivas, Anupama R. Gulur; Mahalingam, Mohana; Visweswariah, Sandhya S.

doi:10.1016/j.biochi.2005.01.017

Cited by 12 publications

(10 citation statements)

References 22 publications

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“…The protein was expressed and purified from E. coli BL21(DE3) cells as described previously for the Rv1647 protein (35). Adenylyl cyclase assays were performed as described previously, and cAMP was measured by radioimmunoassay (36).…”

Section: Methodsmentioning

confidence: 99%

Cyclic AMP in Mycobacteria: Characterization and Functional Role of the Rv1647 Ortholog in Mycobacterium smegmatis

et al. 2008

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Mycobacterial genomes are endowed with many eukaryote-like nucleotide cyclase genes encoding proteins that can synthesize 3,5-cyclic AMP (cAMP). However, the roles of cAMP and the need for such redundancy in terms of adenylyl cyclase genes remain unknown. We measured cAMP levels in Mycobacterium smegmatis during growth and under various stress conditions and report the first biochemical and functional characterization of the MSMEG_3780 adenylyl cyclase, whose orthologs in Mycobacterium tuberculosis (Rv1647) and Mycobacterium leprae (ML1399) have been recently characterized in vitro. MSMEG_3780 was important for producing cAMP levels in the logarithmic phase of growth, since the ⌬MSMEG_3780 strain showed lower intracellular cAMP levels at this stage of growth. cAMP levels decreased in wild-type M. smegmatis under conditions of acid stress but not in the ⌬MSMEG_3780 strain. This was correlated with a reduction in MSMEG_3780 promoter activity, indicating that the effect of the reduction in cAMP levels on acid stress was caused by a decrease in the transcription of MSMEG_3780. Complementation of the ⌬MSMEG_3780 strain with the genomic integration of MSMEG_3780 or the Rv1647 gene could restore cAMP levels during logarithmic growth. The Rv1647 promoter was also acid sensitive, emphasizing the biochemical and functional similarities in these two adenylyl cyclases. This study therefore represents the first detailed biochemical and functional analysis of an adenylyl cyclase that is important for maintaining cAMP levels in mycobacteria and underscores the subtle roles that these genes may play in the physiology of the organism.Cyclic AMP (cAMP) is an important intracellular second messenger that is involved in several signaling pathways in both bacteria and eukaryotes (2, 5). Studies have shown a role for cAMP not only in physiological processes such as catabolite repression and sporulation (16) but also in the regulation of several virulence pathways, e.g., in Pseudomonas, Vibrio, and Candida species (1,17,19,40,41). The synthesis of cAMP is dependent on the activity of adenylyl cyclases, which can be membrane-bound or soluble proteins (7). The largely differing amino acid sequences of nucleotide cyclases allow their classification into six classes, among which the class III nucleotide cyclases have the widest phyletic distribution (7, 37).Adenylyl and guanylyl cyclases of the class III family are proteins that form head-to-tail dimers and generate either two (homodimeric enzymes) or one (heterodimeric enzymes) active site at the dimer interface (20). Nucleotide cyclases catalyze the conversion of ATP or GTP to cAMP or GMP, respectively, in a metal-dependent manner, and biochemical and structural studies have identified specific sequence motifs that are involved in substrate (ATP or GTP) or metal (Mg 2ϩ ) binding (20). In contrast to the limited domain compositions of mammalian nucleotide cyclases, the bacterial counterparts are often fused to a variety of domains, highlighting their ability to be regulated allostericall...

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Section: Methodsmentioning

confidence: 99%

Cyclic AMP in Mycobacteria: Characterization and Functional Role of the Rv1647 Ortholog in Mycobacterium smegmatis

et al. 2008

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“…Comparison of nucleotidyl cyclases has shown that prokaryotic GCs share a close similarity to bacterial ACs. These bacterial ACs in turn resemble mammalian ACs, as shown by several workers [9]–[14]. Ma1120, an adenylyl cyclase present in M. avium shares high sequence similarity with GCs, so this raised the question as to whether Ma1120 could be converted to GC.…”

Section: Introductionmentioning

confidence: 89%

Mutational Analysis Gives Insight into Substrate Preferences of a Nucleotidyl Cyclase from Mycobacterium avium

Syed¹,

Colaςo²,

Misquith³

2014

PLoS ONE

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Mutational, crystallographic and phylogenetic analysis of nucleotidyl cyclases have been used to understand how these enzymes discriminate between substrates. Ma1120, a class III adenylyl cyclase (AC) from Mycobacterium avium, was used as a model to study the amino acid residues that determine substrate preference, by systematically replacing ATP specifying residues with those known to specify GTP. This enzyme was found to possess residual guanylyl cyclase (GC) activity at alkaline pH. Replacement of key residues lysine (101) and aspartate (157) with residues conserved across GCs by site directed mutagenesis, led to a marked improvement in GC activity and a decrease in AC activity. This could be correlated to the presence and strength of the hydrogen bond between the second substrate binding residue (157) and the base of the nucleotide triphosphate. This is substantiated by the fact that the pH optimum is highly dependent on the amino acid residues present at positions 101 and 157.

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“…A functional orthologue was identified in M. avium strain TN104 (cloned and called Ma1120) and M. avium subsp. paratuberculosis (MAP2672) by in silico comparative genomics [29]. Interestingly, Ma1120 is highly active, despite the natural substitution of the transition‐state stabilizing asparagine by alanine (A164) in this protein [29], suggesting a lack of requirement of this residue in catalysis, much like Rv1900c [14].…”

Section: Class III Adenylyl Cyclases In Mycobacteriamentioning

confidence: 99%

“…paratuberculosis (MAP2672) by in silico comparative genomics [29]. Interestingly, Ma1120 is highly active, despite the natural substitution of the transition‐state stabilizing asparagine by alanine (A164) in this protein [29], suggesting a lack of requirement of this residue in catalysis, much like Rv1900c [14]. Indeed, amongst the cyclases whose structures have been elucidated, Ma1120 shares the highest sequence similarity (∼50%) with Rv1900c.…”

Section: Class III Adenylyl Cyclases In Mycobacteriamentioning

confidence: 99%

“…Ma1120 was also the first mycobacterial adenylyl cyclase that was shown to be inhibited by P‐site compounds [29]. The P‐site compound, 2′‐dideoxyadenosine‐3′‐monophosphate had an IC 50 ∼ 50 μM against Ma1120, however, 2′,5′‐dideoxyadenosine‐3′‐triphosphate, with extra phosphate groups on the 3′‐hydroxyl group of ribose, was found to be more potent (IC 50 ∼ 750 nM) [29]. This is similar to the observations with mammalian adenylyl cyclases where multiple phosphate groups on the 3′‐hydroxyl position increase the potency of P‐site compounds [21].…”

Section: Class III Adenylyl Cyclases In Mycobacteriamentioning

confidence: 99%

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Mycobacterial adenylyl cyclases: Biochemical diversity and structural plasticity

Shenoy

Visweswariah

2006

FEBS Letters

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The conversion of adenine and guanine nucleoside triphosphates to cAMP and cGMP is carried out by nucleotide cyclases, which vary in their primary sequence and are therefore grouped into six classes. The class III enzymes encompass all eukaryotic adenylyl and guanylyl cyclase, and several bacterial and archaebacterial cyclases. Mycobacterial nucleotide cyclases show distinct biochemical properties and domain fusions, and we review here biochemical and structural studies on these enzymes from Mycobacterium tuberculosis and related bacteria. We also present an in silico analysis of nucleotide cyclases found in completely sequenced mycobacterial genomes. It is clear that this group of enzymes demonstrates the tinkering in the class III cyclase domain during evolution, involving subtle structural changes that retain the overall catalytic function and fine tune their activities.

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An adenylyl cyclase pseudogene in Mycobacterium tuberculosis has a functional ortholog in Mycobacterium avium

Cited by 12 publications

References 22 publications

Cyclic AMP in Mycobacteria: Characterization and Functional Role of the Rv1647 Ortholog in Mycobacterium smegmatis

Cyclic AMP in Mycobacteria: Characterization and Functional Role of the Rv1647 Ortholog in Mycobacterium smegmatis

Mutational Analysis Gives Insight into Substrate Preferences of a Nucleotidyl Cyclase from Mycobacterium avium

Mycobacterial adenylyl cyclases: Biochemical diversity and structural plasticity

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