A flavin cofactor-binding PAS domain regulates c-di-GMP synthesis in AxDGC2 from Acetobacter xylinum.
The cytoplasmic protein AxDGC2 regulates cellulose synthesis in the obligate aerobe Acetobacter xylinum by controlling the cellular concentration of the cyclic dinucleotide messenger c-di-GMP. AxDGC2 contains a Per-Arnt-Sim (PAS) domain and two putative catalytic domains (GGDEF and EAL) for c-di-GMP metabolism. We found that the PAS domain of AxDGC2 binds a flavin adenine dinucleotide (FAD) cofactor noncovalently. The redox status of the FAD cofactor modulates the catalytic activity of the GGDEF domain for c-di-GMP synthesis, with the oxidized form exhibiting higher catalytic activity and stronger substrate inhibition. The results suggest that AxDGC2 is a signaling protein that regulates the cellular c-di-GMP level in response to the change in cellular redox status or oxygen concentration. Moreover, several residues predicated to be involved in FAD binding and signal transduction were mutated to examine the impact on redox potential and catalytic activity. Despite the minor perturbation of redox potential and unexpected modification of FAD in one of the mutants, none of the single mutations was able to completely disrupt the transmission of the signal to the GGDEF domain, indicating that the change in the FAD redox state can still trigger structural changes in the PAS domain probably by using substituted hydrogen-bonded water networks. Meanwhile, although the EAL domain of AxDGC2 was found to be catalytically inactive toward c-di-GMP, it was capable of hydrolyzing some phosphodiester bond-containing nonphysiological substrates. Together with the previously reported oxygen-dependent activity of the homologous AxPDEA1, the results provided new insight into relationships among oxygen level, c-di-GMP concentration, and cellulose synthesis in A. xylinum.
Division of Chemical Biology and Biotechnology, School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551.
Yaning
Y
Qi
Feng
F
Rao
Zhen
Z
Luo
Zhao-Xun
ZX
Liang
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000502:physiology*; D002482:Cellulose / Q000096:biosynthesis; D002851:Chromatography, High Pressure Liquid; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000096:biosynthesis; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics / Q000502:physiology; D041822:Gluconacetobacter xylinus / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010084:Oxidation-Reduction; D010802:Phylogeny; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology
D001426:Bacterial Proteins; D005182:Flavin-Adenine Dinucleotide; C062025:bis(3',5')-cyclic diguanylic acid; D002482:Cellulose; D006152:Cyclic GMP