Rhizobium meliloti adenylate cyclase is related to eucaryotic adenylate and guanylate cyclases

Beuve, Annie; Boesten, Bert; Crasnier, Martine; Danchin, Antoine; O’Gara, Fergal

doi:10.1128/jb.172.5.2614-2621.1990

Cited by 49 publications

(25 citation statements)

References 39 publications

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“…32). The proteins were expressed in the Escherichia coli strain TP2000, which is incapable of producing cAMP (42). The expression of VC1C2 and VC1aC2, cell lysis and assays were performed as described previously (32).…”

Section: Methodsmentioning

confidence: 99%

Inhibition by Calcium of Mammalian Adenylyl Cyclases

Guillou

Nakata

Cooper

1999

Journal of Biological Chemistry

108

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Ca2؉ regulates mammalian adenylyl cyclases in a type-specific manner. Stimulatory regulation is moderately well understood. By contrast, even the concentration range over which Ca 2؉ inhibits adenylyl cyclases AC5 and AC6 is not unambiguously defined; even less so is the mechanism of inhibition. In the present study, we compared the regulation of Ca 2؉ -stimulable and Ca 2؉ -inhibitable adenylyl cyclases expressed in Sf9 cells with tissues that predominantly express these activities in the mouse brain. Soluble forms of AC5 containing either intact or truncated major cytosolic domains were also examined. All adenylyl cyclases, except AC2 and the soluble forms of AC5, displayed biphasic Ca 2؉ responses, suggesting the presence of two Ca 2؉ sites of high (ϳ0.2 M) and low affinity (ϳ0.1 mM). With a high affinity, Ca 2؉ (i) stimulated AC1 and cerebellar adenylyl cyclases, (ii) inhibited AC6 and striatal adenylyl cyclase, and (iii) was without effect on AC2. With a low affinity, Ca 2؉ inhibited all adenylyl cyclases, including AC1, AC2, AC6, and both soluble forms of AC5. The mechanism of both high and low affinity inhibition was revealed to be competition for a stimulatory Mg 2؉ site(s). A remarkable selectivity for Ca 2؉ was displayed by the high affinity site, with a K i value of ϳ0.2 M, in the face of a 5000-fold excess of Mg 2؉. The present results show that high and low affinity inhibition by Ca 2؉ can be clearly distinguished and that the inhibition occurs type-specifically in discrete adenylyl cyclases. Distinction between these sites is essential, or quite spurious inferences may be drawn on the nature or location of high affinity binding sites in the Ca 2؉ -inhibitable adenylyl cyclases.Profound physiological significance derives from the regulation of adenylyl cyclase by Ca 2ϩ

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Section: Methodsmentioning

confidence: 99%

Inhibition by Calcium of Mammalian Adenylyl Cyclases

Guillou

Nakata

Cooper

1999

Journal of Biological Chemistry

108

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“…The TP2000 strain of E. coli, which does not express AC (22,23), was used. Cells transformed with the plasmid constructs encoding C1-C2 or C1a-C2 forms of ACV were grown in Luria's broth containing ampicillin (50 g͞ml) at 37ЊC until they reached an OD 600 of 0.4.…”

Section: Constructs For Expression Of Recombinant Proteinsmentioning

confidence: 99%

Characterization of soluble forms of nonchimeric type V adenylyl cyclases

Scholich

Barbier

Mullenix

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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Type V adenylyl cyclase (ACV) belongs to the family of Ca 2+ - inhibited cyclases. We have generated two soluble forms of the enzyme containing the C1 or C1a region (which lacks the C-terminal 112 amino acids) linked to the C2 domain and compared their regulation with the full-length ACV. All three forms of ACV were stimulated by the α subunit of the stimulatory G protein G s (G sα ) and forskolin. However, the synergistic stimulation by both these activators was markedly enhanced in the soluble enzymes. Moreover, the α subunit of the inhibitory G protein G i (G iα ) inhibited all forms of the enzyme, indicating that the regions for G sα and G iα interaction are preserved in the soluble forms. Ca 2+ inhibited forskolin-stimulated adenylyl cyclase (AC) activity of the full-length and C1-C2 forms of ACV but did not alter the activity of the C1a-C2 form. Maximal stimulation of AC activity by combination of G sα and forskolin obliterated the Ca 2+ -mediated inhibition of the full-length and C1-C2 forms of ACV. In 45 Ca 2+ overlay experiments, the C1-C2 but not the C1a-C2 soluble ACV bound Ca 2+ . Moreover, proteins corresponding to the C1a and C2 domains did not bind calcium. On the other hand, the proteins corresponding to C1 and its C-terminal 112 amino acids (C1b) bound 45 Ca 2+ . To our knowledge, this is the first report of nonchimeric soluble forms of AC in which regulation by G sα and G iα is preserved. Moreover, we demonstrate that the 112 amino acid C1b region of ACV is responsible for the binding of Ca 2+ and inhibition of enzyme activity.

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“…More than 30 different adenylyl cyclases and guanylyl cyclases share the common homologous cytoplasmic domains, designated as the cyclase domains (1). For the functional enzyme, the cyclase domains can work as the homo-oligomer, such as adenylyl cyclases from yeast, Trypanosome, Dictyostelium (ACG), Leishmania, Brevibacterium, and Rhizobium (23)(24)(25)(26)(27)(28)(29)(30). The cyclase domains can also exist as an obligatory heterodimer for enzymatic activity such as the C 1 and C 2 domains from type I to type IX adenylyl cyclases, Drosophila rutabaga, and Dictyostelium ACA, and the ␣ and ␤ subunits of soluble guanylyl cyclases (22,23,(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46).…”

Section: Isolation and Analysis Of Loss Of Function Ic 1 Iic 2 Mutants-mentioning

confidence: 99%

“…Third are the peripheral membrane proteins from yeast (Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Saccharomyces klyuyveri) (26 -28). Fourth are the soluble enzymes from the bacteria, Brevibacterium liquefaciens (Bl AC) and Rhizobium meliloti (Rm AC) (29,30). 2 The abbreviations used are: G protein, guanine nucleotide binding regulatory protein; G s␣ , the ␣ subunit of the G protein that stimulates adenylyl cyclase; Fsk, forskolin; IC 1 protein, C 1 domain of type I adenylyl cyclase; IIC 2 protein, C 2 domain of type II adenylyl cyclase; GTP␥S, guanosine-5Ј-[␥-thio]triphosphate; aa, amino acid(s); 2Ј-D-3Ј-AMP, 2Јde-oxyadenosine 3Ј-monophosphate; kbp, kilobase pair; IPTG, isopropyl-1-thio-␤-D-galactopyranoside; LB, Luria broth; CMV, cytomegalovirus; IBMX, isobutylmethylxanthine.…”

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confidence: 99%

The Conserved Asparagine and Arginine Are Essential for Catalysis of Mammalian Adenylyl Cyclase

Yan

Huang

Shaw

et al. 1997

Journal of Biological Chemistry

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Mammalian adenylyl cyclases have two homologous cytoplasmic domains (C 1 and C 2 ), and both domains are required for the high enzymatic activity. Mutational and genetic analyses of type I and soluble adenylyl cyclases suggest that the C 2 domain is catalytically active and the C 1 domain is not; the role of the C 1 domain is to promote the catalytic activity of the C 2 domain. Two amino acid residues, Asn-1025 and Arg-1029 of type II adenylyl cyclase, are conserved among the C 2 domains, but not among the C 1 domains, of adenylyl cyclases with 12 putative transmembrane helices. Mutations at each amino acid residue alone result in a 30 -100-fold reduction in K cat of adenylyl cyclase. However, the same mutations do not affect the K m for ATP, the half-maximal concentration (EC 50 ) for the C 2 domain of type II adenylyl cyclase to associate with the C 1 domain of type I adenylyl cyclase and achieve maximal enzyme activity, or the EC 50 for forskolin to maximally activate enzyme activity with or without G s␣ . This indicates that the mutations at these two residues do not cause gross structural alteration. Thus, these two conserved amino acid residues appear to be crucial for catalysis, and their absence from the C 1 domains may account for its lack of catalytic activity. Mutations at both amino acid residues together result in a 3,000-fold reduction in K cat of adenylyl cyclase, suggesting that these two residues have additive effects in catalysis. A second site suppressor of the Asn-1025 to Ser mutant protein has been isolated. This suppressor has 17-fold higher activity than the mutant and has a Pro-1015 to Ser mutation.The activity of mammalian adenylyl cyclase, the enzyme that converts ATP to cAMP, is the key step in modulating intracellular cAMP concentration in response to extracellular stimulation by hormones, neurotransmitters, and odorants. Nine isoforms of mammalian adenylyl cyclases have been cloned to date, and they belong to a rapid expanding cyclase family that includes class III adenylyl cyclases 1 and guanylyl cyclases (1).All nine isoforms have a similar structure, including two intensely hydrophobic domains (M 1 and M 2 ) and two 40-kDa cytoplasmic domains (C 1 and C 2 ). The two cytoplasmic domains contain sequences (C 1a and C 2a ) that are homologous to each other and to class III adenylyl cyclases and guanylyl cyclases. Each isoform of adenylyl cyclase not only has distinct patterns of tissue expression but also has unique responses to extracellular and intracellular stimuli (1-3). In common, each adenylyl cyclase can be activated by the G protein 2 ␣ subunit, designated as G s␣ , and each can be inhibited by certain adenosine analogues (P-site inhibitors). However, there are several subtype-specific regulators, including forskolin, G protein ␤␥ subunits, the ␣ subunit of G i , G o , and G z , Ca 2ϩ ion, Ca 2ϩ -calmodulin, Ca 2ϩ -calcineurin, cAMP-dependent protein kinase, and PKC (for review, see Refs. 1-3). Certain ones of these regulators (e.g. ␤␥) can be either stimulatory or inhibitory...

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Rhizobium meliloti adenylate cyclase is related to eucaryotic adenylate and guanylate cyclases

Cited by 49 publications

References 39 publications

Inhibition by Calcium of Mammalian Adenylyl Cyclases

Inhibition by Calcium of Mammalian Adenylyl Cyclases

Characterization of soluble forms of nonchimeric type V adenylyl cyclases

The Conserved Asparagine and Arginine Are Essential for Catalysis of Mammalian Adenylyl Cyclase

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