## 3 Results and Discussion
### BLUF Modules in Association with the Effector Domains
#### 3.3.3 Cyclase Homology Domain (CHD)  
The cyclase homology domains (CHDs) are the catalytic domains of eukaryotic and prokaryotic nucleotidlyclyclases, i.e., adenylyl cyclases (ACs) and guanylyl cyclases (GCs), which belong to the evolutionary diverse class III nucleotidlycyclases. CHDs are reported as three different structural forms, i.e., heterodimers (mammalian CHDs), pseudoheterodimers (metazoan CHDs) and homodimers (bacterial and protozoan CHDs) [85]. Heterodimeric and pseudoheterodimeric CHDs have a single catalytic pocket sharing catalytic amino acid residues at the dimer interface, while homodimeric CHDs have two separate catalytic pockets, each of which contributes the CHD determinant [85]. In spite of having two potential catalytic pockets, several enzymes with homodimeric CHDs (for example, eukaryotic class III nucleotidylcyclases) may have only one catalytically competent site [86]. Although CHDs are structurally diverse, all of them have a conserved structural component (i.e., a helical region) mutating which compromises the stability and active dimeric conformation of a protein [87]. All CHDs have a common catalytic mechanism in which they require two magnesium or manganese ions to bind a polyphosphate group of the nucleotide, followed by nucleophile activation.  
Most CHDs, except a few (Rv1359 from _M. tuberculosis_), are reported to exist in combination with different regulatory modules, thus making it possible to perceive the variety of signals and the regulation of the intracellular cAMP generation [85]. In the present communication, we have selected three such proteins, with the BLUF domain in combination with the CHD domain, having the accession numbers EHQ08139.1 (from _Leptonema illini_ DSM 21528), Q8S9F2.1 (from _Euglena gracilis_) and XP_013758351.1 (from _Thecamonas trahens_ ATCC 50062) (Table 1). In _Euglena gracilis_, CHDs (adenylyl cyclases) occur in combination with the BLUF domain, with an overall domain arrangement of BLUF1 CHD1 BLUF2 CHD2, where the flavin chromophore senses blue light and stimulates the adenylyl cyclase activity [35]. Furthermore, we have performed a alignment of sequences representing the CHD domain from selected proteins against the progression of the well-characterized template CHD domain of bPAC (Figure S2). The multiple sequence alignment revealed that active site residues involved in the nucleotide binding (i.e., Asn257-His266, Lys263-Met264 (forming the $4 AC- $5 AC tongue), Gly259-Asn178 (forming the $2 AC helix) and Lys263-Thr196, Asp265-Phe198, and His266-Lys197 (forming the $2 AC- $3 AChairpin) [33]) are highly conserved among all three selected BLUF-CHD domain-containing proteins (Figure S2). In PACs, under dark conditions, the orientation and arrangement of an individual amino acid in the active site (i.e., Thr267 and Lys197 (bound to Phe198)) render the conformation of AC inactive. On the other hand, upon illumination, the change in the interaction between Asn25 and His266 resulted inthe correct orientation of Thr267 required for the communication with the adenine base; additionally, the Lys197 is detached from Phe198, thus providing space for the adenine base to enter the active site more deeply [33]. We also performed a protein-protein interaction analysis, which revealed the possible interacting partners for CHD domain-containing proteins, which range from phosphodiesterase, the RNA polymerase subunit \(\beta\), and the DNA helicase to another adenylate cyclase/guanylate cyclase associated with the GAF and PAS/PAC sensor (Figure S7b and Table S1).