Jürgen U. Linder

Class III adenylyl cyclases are the most abundant type of cyclic AMP-producing enzymes. The adjustment of the cellular levels of this second messenger is achieved by a variety of regulatory mechanisms which couple signals to adenylyl cyclase activity. Because of the divergent nature of stimuli which impinge on these enzymes, highly individualized class III adenylyl cyclases have evolved in metazoans, eukaryotic unicells and bacteria. Regulation usually exploits the dimeric structure of the catalyst, whose active centres form at the dimer interface. The fold of the catalytic domains and the basic catalytic mechanisms are similar in all class III adenylyl cyclases, and substrate binding generally closes the active site by an induced-fit mechanism. Regulatory inputs can result in dramatic rearrangements of the catalytic domains within the dimer, which often are based on rotational movements.

The second messengers cAMP and cGMP are of central importance in signal transduction pathways. To assure pathway specificity adenylyl and guanylyl cyclases are highly selective for their substrates, ATP and GTP, respectively. The universal class III cyclases are equipped with a variety of purine‐binding modes, which have been identified by structure determination and mutagenesis. Most selection mechanisms rely on a pair of residues which form hydrogen bonds to N1 and the N6‐amino or O6‐keto group of adenine and guanine, respectively. Furthermore, selection is supported by hydrogen bonds involving the peptide backbone and by constraints imposed by hydrophobic side‐chains.

Class III adenylyl cyclases contain catalytic and regulatory domains, yet structural insight into their interactions is missing. We show that the mycobacterial adenylyl cyclase Rv1264 is rendered a pH sensor by its N-terminal domain. In the structure of the inhibited state, catalytic and regulatory domains share a large interface involving catalytic residues. In the structure of the active state, the two catalytic domains rotate by 55° to form two catalytic sites at their interface. Two α helices serve as molecular switches. Mutagenesis is consistent with a regulatory role of the structural transition, and we suggest that the transition is regulated by pH.

Rv1900c, a Mycobacterium tuberculosis adenylyl cyclase, is composed of an N‐terminal α/β‐hydrolase domain and a C‐terminal cyclase homology domain. It has an unusual 7% guanylyl cyclase side‐activity. A canonical substrate‐defining lysine and a catalytic asparagine indispensable for mammalian adenylyl cyclase activity correspond to N342 and H402 in Rv1900c. Mutagenic analysis indicates that these residues are dispensable for activity of Rv1900c. Structures of the cyclase homology domain, solved to 2.4 Å both with and without an ATP analog, form isologous, but asymmetric homodimers. The noncanonical N342 and H402 do not interact with the substrate. Subunits of the unliganded open dimer move substantially upon binding substrate, forming a closed dimer similar to the mammalian cyclase heterodimers, in which one interfacial active site is occupied and the quasi‐dyad‐related active site is occluded. This asymmetry indicates that both active sites cannot simultaneously be catalytically active. Such a mechanism of half‐of‐sites‐reactivity suggests that mammalian heterodimeric adenylyl cyclases may have evolved from gene duplication of a primitive prokaryote‐type cyclase, followed by loss of function in one active site.

The genes Rv1318c, Rv1319c, Rv1320c and Rv3645 of Mycobacterium tuberculosis are predicted to code for four out of 15 adenylyl cyclases in this pathogen. The proteins consist of a membrane anchor, a HAMP region and a class IIIb adenylyl cyclase catalytic domain. Expression and purification of the isolated catalytic domains yielded adenylyl cyclase activity for all four recombinant proteins. Expression of the HAMP region fused to the catalytic domain increased activity in Rv3645 21-fold and slightly reduced activity in Rv1319c by 70%, demonstrating isoform-specific effects of the HAMP domains. Point mutations were generated to remove predicted hydrophobic protein surfaces in the HAMP domains. The mutations further stimulated activity in Rv3645 eight-fold, whereas the effect on Rv1319c was marginal. Thus HAMP domains can act directly as modulators of adenylyl cyclase activity. The modulatory properties of the HAMP domains were confirmed by swapping them between Rv1319c and Rv3645. The data indicate that in the mycobacterial adenylyl cyclases the HAMP domains do not display a uniform regulatory input but instead each form a distinct signaling unit with its adjoining catalytic domain.

The class IIIa adenylyl cyclase (AC) Rv1625c from Mycobacterium tuberculosis forms homodimers with two catalytic centres, whereas the Paramecium guanylyl and mammalian ACs operate as pseudoheterodimers with one catalytic centre. The functional and structural relationship of the catalytic domains of these related class III cyclases was investigated. Point mutations introduced into Rv1625c to engineer a forskolin‐binding pocket created a single heterodimeric catalytic centre, yet did not result in forskolin activation. Chimerization of these Rv1625c point mutants with corresponding mammalian AC domains was impossible. However, it was successful using a complemental Paramecium guanylyl cyclase domain and resulted in an AC. The data signify a divergence of structural and functional evolution in class III Acs.

The gene cyaB1 from the cyanobacterium Anabaena sp. PCC 7120 codes for a protein consisting of two N‐terminal GAF domains (GAF‐A and GAF‐B), a PAS domain and a class III adenylyl cyclase catalytic domain. The catalytic domain is active as a homodimer, as demonstrated by reconstitution from complementary inactive point mutants. The specific activity of the holoenyzme increased exponentially with time because the product cAMP activated dose dependently and nucleotide specifically (half‐maximally at 1 μM), identifying cAMP as a novel GAF domain ligand. Using point mutants of either the GAF‐A or GAF‐B domain revealed that cAMP activated via the GAF‐B domain. We replaced the cyanobacterial GAF domain ensemble in cyaB1 with the tandem GAF‐A/GAF‐B assemblage from the rat cGMP‐stimulated phosphodiesterase type 2, and converted cyaB1 to a cGMP‐stimulated adenylyl cyclase. This demonstrated the functional conservation of the GAF domain ensemble since the divergence of bacterial and eukaryotic lineages >2 billion years ago. In cyanobacteria, cyaB1 may act as a cAMP switch to stabilize committed developmental decisions.