A LOV-domain-mediated blue-light-activated adenylate (adenylyl) cyclase from the cyanobacterium Microcoleus chthonoplastes PCC 7420

Abstract

Genome screening of the cyanobacterium Microcoleus chthonoplastes PCC 7420 identified a gene encoding a protein (483 amino acids, 54.2 kDa in size) characteristic of a BL (blue light)-regulated adenylate (adenylyl) cyclase function. The photoreceptive part showed signatures of a LOV (light, oxygen, voltage) domain. The gene product, mPAC (Microcoleus photoactivated adenylate cyclase), exhibited the LOV-specific three-peaked absorption band (λmax=450 nm) and underwent conversion into the photoadduct form (λmax=390 nm) upon BL-irradiation. The lifetime for thermal recovery into the parent state was determined as 16 s at 20°C (25 s at 11°C). The adenylate cyclase function showed a constitutive activity (in the dark) that was in-vitro-amplified by a factor of 30 under BL-irradiation. Turnover of the purified protein at saturating light and pH 8 is estimated to 1 cAMP/mPAC per s at 25°C (2 cAMP/mPAC per s at 35°C). The lifetime of light-activated cAMP production after a BL flash was ~14 s at 20°C. The temperature optimum was determined to 35°C and the pH optimum to 8.0. The value for half-maximal activating light intensity is 6 W/m2 (at 35°C). A comparison of mPAC and the BLUF (BL using FAD) protein bPAC (Beggiatoa PAC), as purified proteins and expressed in Xenopus laevis oocytes, yielded higher constitutive activity for mPAC in the dark, but also when illuminated with BL.

Genomic Survey and Biochemical Analysis of Recombinant Candidate Cyanobacteriochromes Reveals Enrichment for Near UV/Violet Sensors in the Halotolerant and Alkaliphilic Cyanobacterium Microcoleus IPPAS B353

“…The natural habitat of Microcoleus B353 therefore would expose this organism to a wide range of solar radiation unfiltered by other photoautotrophs. The Microcoleus B353 genome also lacks UV-B sensing UVR8 (63), near-UV intensity sensor SyUirS (22), and flavin-based blue light sensing photoreceptors with the lightoxygen-voltage domain, such as MPAC (ZP_05024462.1) from Microcoleus chthonoplastes PCC 7420 (64,65) or the BLUF domain, such as Slr1694 from Synechocystis PCC 6803 (66,67). It is therefore reasonable that such short wavelength near-UV and violet sensing CBCRs would provide an adaptive advantage to living in high light environments.…”

Photoreceptor engineering

“…By that token, in an ideal scenario, a natural photoreceptor exists that already exerts the demanded light-regulated biological activity and that can hence be used in optogenetics without any modification. Notable representatives are the light-gated channelrhodopsins (Nagel et al, 2002, 2003), certain animal rhodopsins (Oh et al, 2010; Spoida et al, 2014) and several light-activated nucleotide cyclases based on BLUF, LOV and rhodopsin photosensors (Schröder-Lang et al, 2007; Ryu et al, 2010; Stierl et al, 2011; Raffelberg et al, 2013; Avelar et al, 2014). Given the vast amounts of genome data (becoming) available, additional protein architectures of immediate optogenetic utility may be discovered in future (Figure 6).…”

“…Depending upon application, overall activity has to be adjusted to certain ranges to achieve relevant light-induced effects. If several photoreceptor systems with congruent functionality are available, as for light-activated adenylate cyclases (Schröder-Lang et al, 2007; Ryu et al, 2010; Stierl et al, 2011; Raffelberg et al, 2013; Avelar et al, 2014), one may select according to the specific activity of these systems. As outlined above, photoreceptors can be integrated into downstream signaling cascades so as to modulate or amplify the light effect.…”

Biology of Light-Sensing Proteins in Plants and Microorganisms

“…LOV-histidine kinases identifi ed in bacteria transduce light signals as sensors of the bacterial twocomponent system, inducing various biological responses (Swartz et al 2007 ). Recently, a light-activated adenylyl cyclase containing a LOV domain in a cyanobacterium has been reported (Raffelberg et al 2013 ). …”