Adenylate cyclases (ACs), much like guanylate cyclases (GCs), are increasingly recognized as essential parts of many plant processes including biotic and abiotic stress responses. In order to identify novel ACs, we have applied a search motif derived from experimentally tested GCs and identified a number of Arabidopsis thaliana candidates including a clathrin assembly protein (AT1G68110; AtClAP). AtClAP contains a catalytic centre that can complement the AC-deficient mutant cyaA in E. coli, and a recombinant AtClAP fragment (AtClAP^261–379) can produce cyclic adenosine 3′,5′ monophosphate (cAMP) from adenosine triphosphate (ATP) in vitro. Furthermore, an integrated analysis of gene expression and expression correlation implicate cAMP in pathogen defense and in actin cytoskeletal remodeling during endocytic internalization.

It is increasingly clear that plant genomes encode numerous complex multidomain proteins that harbor functional adenylyl cyclase (AC) centers. These AC containing proteins have well-documented roles in development and responses to the environment. However, it is only for a few of these proteins that we are beginning to understand the intramolecular mechanisms that govern their cellular and biological functions, as detailed characterizations are biochemically and structurally challenging given that these poorly conserved AC centers typically constitute only a small fraction (<10%) of complex plant proteins. Here, we offer fresh perspectives on their seemingly cryptic activities specifically showing evidence for the presence of multiple functional AC centers in a single protein and linking their catalytic strengths to the Mg²⁺/Mn²⁺-binding amino acids. We used a previously described computational approach to identify candidate multidomain proteins from Arabidopsis thaliana that contain multiple AC centers and show, using an Arabidopsis leucine-rich repeat containing protein (TAIR ID: At3g14460; AtLRRAC1) as example, biochemical evidence for multienzymatic activities. Importantly, all AC-containing fragments of this protein can complement the AC-deficient mutant cyaA in Escherichia coli, while structural modeling coupled with molecular docking simulations supports catalytic feasibility albeit to varying degrees as determined by the frequency of suitable substrate binding poses predicted for the AC sites. This statistic correlates well with the enzymatic assays, which implied that the greatly reduced AC activities is due to the absence of the negatively charged [DE] amino acids previously assigned to cation-, in particular Mg²⁺/Mn²⁺-binding roles in ACs.

Fast-growing and highly adaptable avian birds such as quail (Coturnix coturnix) possess great potential to meet the growing demand for animal protein by the rapidly increasing human population, and would contribute immensely to global food production and nutritional security. However, overreliance on conventional protein sources such as fish and soybean meals during the formulation of quail diets is economically and environmentally unsustainable. Alternatively, insect-based protein sources such as Gonimbrasia belina, commonly known as mopane worm (MW), can be used to increase quail production due to their high biological value and low feed-food competition. Indeed, MW is highly nutritious, with an average protein content of 55% and a well-balanced amino acid profile. Thus, its incorporation in quail diets could provide great potential to alleviate nutritional deficiencies in quail production and allow for their sustainable intensification. However, there are limited studies on the effect of partial or complete replacement of conventional protein sources with mopane worm meal (MWM) in quail diets. This paper reviews the nutritional profile and use of the MW as a protein source, as well as its potential future prospects in poultry diets. Finally, we postulate that mass production of this insect-based protein source and its sustainability would be an inventive strategy to develop a profitable quail business.

Background: Second messengers play a key role in linking environmental stimuli to cellular responses. One such messenger, 3′,5′-cyclic adenosine monophosphate (cAMP) generated by adenylyl cyclase (AC), has long been established as an essential signaling molecule in many physiological processes of higher plants, including growth, development, and stress response. Very few ACs have been identified in plants so far, so more must be sought. Objective: To test the probable AC activity of an Arabidopsis MEE (AtMEE) protein and infer its function bioinformatically. Methods: A truncated version of the AtMEE protein (encoded by At2g34780 gene) harboring the annotated AC catalytic center (AtMEE-AC) was cloned and expressed in BL21 Star pLysS Escherichia coli cells followed by its purification using the nickel-nitriloacetic acid (Ni-NTA) affinity system. The purified protein was tested for its probable in vitro AC activity by enzyme immunoassay. The AtMEE-AC protein was also expressed in the SP850 mutant E. coli strain, followed by an assessment (visually) of its ability to complement the AC-deficiency (cyaA mutation) in this mutant. Finally, the AtMEE protein was analyzed bioinformatically to infer its probable biological function(s). Results: AtMEE is an AC molecule whose in vitro activity is Mn²⁺-dependent and positively modulated by NaF. Moreover, AtMEE is capable of complementing the AC-deficiency (cyaA) mutation in the SP850 mutant strain. AtMEE is primarily involved in embryo development and also specifically expressed in response to abiotic stress via the MYB expression core motif signaled by cAMP. Conclusion: AtMEE is an AC protein whose functions are associated with embryo development and response to abiotic stress.

Adenylyl cyclase (AC) is an enzyme that catalyses the formation of the second messenger molecule, 3′,5′-cyclic adenosine monophosphate (cAMP) from 5′-adenosine triphosphate (ATP). cAMP, in turn, regulates key physiological processes such as cell division, growth, reproduction, development and response to stress. However, while cAMP is increasingly becoming an important signalling molecule in higher plants, the identification of plant ACs has somewhat remained so slow. In Arabidopsis thaliana alone, only twelve ACs have so far been identified, yet considering the number and diverse nature of processes known to be cAMP-dependent in this plant, these identified ACs are still very much few to account for that. Notably, an additional protein in this plant, termed linker histone-like (AtLHL) protein (encoded by the At3g18035 gene), is annotated to be an AC as result of it containing a putative centre identical to the one commonly found in the other twelve previously confirmed Arabidopsis ACs. In addition, AtLHL is mostly involved in a number of key cellular processes such as heterochromatin formation, DNA repair, apoptosis, embryogenesis, reproduction and disease resistance that are all modulated by cAMP, yet AtLHL still remains unconfirmed as an AC. As a result, we targeted this protein in this study to determine if it is indeed an AC. To begin with, we used computational analysis to assess the 3-dimensional (3D) structure of AtLHL and found that its AC centre is solvent-exposed, amenable to the unhindered access of ATP as a substrate for catalysis. Next, we cloned, partially expressed and affinity purified a truncated version of this protein (AtLHL^301−480), followed by assessment of its probable AC activity. Through enzyme immunoassay and mass spectrometry, we showed that the recombinant AtLHL^301−480 protein can generate cAMP from ATP in vitro in a manganese-dependent manner that is enhanced by calcium and hydrogen carbonate. In addition, we also showed that the recombinant AtLHL^301−480 protein can complement AC-deficiency (cyaA mutation) in SP850 cells when expressed in this mutant Escherichia coli host strain. We then used electrochemistry to evaluate the molecular interaction of AtLHL^301−480 with its co-factors and modulators during catalysis and activation, respectively, and found that the protein does this physically. This observation then prompted us to specifically search for the presence (and possibly frequency) of calcium-binding sites within the AtLHL protein. Through in silico analysis and bioinformatic studies, a single binding site in form of a 16-residue calmodulin-binding sequence was predicted. Lastly, we then evaluated the reaction kinetics of AtLHL^301−480 and determined that the protein has a K_m constant of 0.7 mM and a V_max constant of 9.2 fmol/min/μg protein. All in all, our study provided adequate evidence in a multi-faceted manner that LHL from A. thaliana is a bona fide AC, whose activity might be involved in control and molecular regulation of the various functions of this protein in this plant.