Photodynamics of photo-activated BLUF coupled Endonuclease III mutant RmPAE from mesophilic, pigmented bacterium Rubellimicrobium mesophilum strain MSL-20T

The pink to light reddish-pigmented bacterium Rubellimicrobium mesophilum strain MSL-20T contains a BLUF coupled endonuclease III of unknown function. A purified recombinant triple mutated sample of the BLUF coupled endonuclease III (F5Y, N27H, A87W) named RmPAE (Rubellimicrobium mesophilum Photo-Activated Endonuclease) was produced and characterized by optical spectroscopic methods. The BLUF domain photo-cycling dynamics occurred with high efficient blue-light induced signaling state formation (quantum yield of signaling state formation φs ≈ 0.6), small spectral red-shift (δλs,r ≈ 5.4 nm), and slow thermal activated dark recovery to the receptor state (τrec ≈ 20 min at room temperature). An apparent RmPAE melting temperature of ϑm ≈ 63 °C was determined by stepwise sample heating and absorption spectrum analysis. The photo-degradation of RmPAE in the signaling state was determined by prolonged intense blue-light exposure. An irreversible flavin photo-degradation occurred with quantum yield of φD ≈ 2.6×10-5. Schemes of the photo-cycling and the photo-degradation dynamics are presented. Engineered RmPAE may find application as light guided DNA cutter in optogenetic applications

Photo-dynamics of photoactivated adenylyl cyclase TpPAC from the spirochete bacterium Turneriella parva strain HT

The photoactivated adenylyl cyclase TpPAC from the spirochete bacterium Turneriella parva was synthesized and the purified recombinant protein was characterized by biochemical and optical spectroscopic methods. TpPAC consists of a BLUF domain (BLUF = Blue Light sensor Using Flavin) and an adenylyl cyclase homology domain (CHD). A light induced cAMP cyclase activity of ≈ 53.3 nmol mg− 1 min− 1 was measured while in the dark the cyclase activity was approximately a factor of 240 lower. The photo-cycling dynamics of the BLUF domain of TpPAC was studied by absorption spectra, fluorescence quantum distribution, and fluorescence lifetime measurements. The quantum efficiency of BLUF domain signaling state formation was found to be ϕs ≈ 0.59. A three-component exponential recovery of the signaling state to the receptor state was observed with the time constants τrec,1 = 4.8 s, τrec,2 = 34.2 s, and τrec,3 = 293 s at 21.3 °C. The protein thermal stability was studied by stepwise sample heating and cooling. An apparent TpPAC melting temperature of ϑm ≈ 46 °C was determined. The photo-degradation of TpPAC in the signaling state was studied by prolonged intense light exposure at 455 nm. An irreversible flavin photo-degradation was observed with quantum yield ϕD ≈ 8.7 × 10− 6

Biochemical characterization of photoactivated adenylyl cyclase from Naegleria gruberi

Blue light sensors using FAD (BLUF) domains are flavin based blue light photoreceptors. The BLUF domains are often fused with various effector domains. BLUF domain coupled with adenylyl cyclase domain is known as photoactivated adenylyl cyclase (PAC). Naegleria gruberi genome database analysis revealed the presence of four PACs. Each of the photoactivated adenylyl cyclases from Naegleria gruberi (NgPACs) is composed of a BLUF domain and an adenylyl cyclase domain. Light regulated enzymatic activity of recombinant NgPAC1 protein was assayed in dark and after blue light irradiation by measuring the cAMP level. Experimental results showed that the NgPAC1 protein exhibits light regulated cyclase activity. In this report, we have also demonstrated that the recombinant NgPAC1 exits as an oligomer in solution

Photo-dynamics of BLUF domain containing adenylyl cyclase NgPAC3 from the amoeboflagellate Naegleria gruberi NEG-M strain

The absorption and emission spectroscopic behavior of the photo-activated adenylyl cyclase NgPAC3 from the amoeboflagellate Naegleria gruberi NEG-M strain was studied. The flavin cofactor was found to be partly fully oxidized and partly fully reduced. The typical BLUF domain (BLUF = Blue Light sensor Using Flavin) oxidized flavin absorption photo-cycle dynamics with about 14 nm flavin absorption red-shift in the signaling state was observed. The quantum efficiency of signaling state formation was determined to be ϕs = 0.66 ± 0.03. A bi-exponential signaling state recovery to the dark-adapted receptor state was observed with the time constants τrec,f = 275 s and τrec,sl = 45 min. The thermal irreversible protein unfolding was studied and an apparent protein melting temperature of ϑm ≈ 50 °C was found. The photodynamic behavior of NgPAC3 is compared with the behavior of the previously investigated photo-activated cyclases NgPAC1 (nPAC) and NgPAC2 from the same N. gruberi NEG-M strain. Purified recombinant NgPAC3 showed light-gated adenylate cyclase activity upon illumination with blue light. Its cyclase activity is compared with those of NgPAC1 and NgPAC2