Biochemical characterization of the carotenoid 1,2-hydratases (CrtC) from Rubrivivax gelatinosus and Thiocapsa roseopersicina

Two carotenoid 1,2-hydratase (CrtC) genes from the photosynthetic bacteria Rubrivivax gelatinosus and Thiocapsa roseopersicina were cloned and expressed in Escherichia coli in an active form and purified by affinity chromatography. The biochemical properties of the recombinant enzymes and their substrate specificities were studied. The purified CrtCs catalyze cofactor independently the conversion of lycopene to 1-HO- and 1,1′-(HO)2-lycopene. The optimal pH and temperature for hydratase activity was 8.0 and 30°C, respectively. The apparent Km and Vmax values obtained for the hydration of lycopene were 24 μM and 0.31 nmol h−1 mg−1 for RgCrtC and 9.5 μM and 0.15 nmol h−1 mg−1 for TrCrtC, respectively. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis revealed two protein bands of 44 and 38 kDa for TrCrtC, which indicate protein processing. Both hydratases are also able to convert the unnatural substrate geranylgeraniol (C20 substrate), which functionally resembles the natural substrate lycopene

Study Towards Carotenoid 1,2-Hydratase and Oleate Hydratase as Novel Biocatalysts

BiotechnologyApplied Science

Identification of catalytically important residues of the carotenoid 1,2-hydratases from Rubrivivax gelatinosus and Thiocapsa roseopersicina

Carotenoid 1,2-hydratases (CrtC) catalyze the selective addition of water to an isolated carbon–carbon double bond. Although their involvement in the carotenoid biosynthetic pathway is well understood, little is known about the mechanism by which these hydratases transform carotenoids such as lycopene into the corresponding hydroxyl compounds. Key residues were identified at positions His239, Trp241, Tyr266, and Asp268 in CrtC from Rubrivivax gelatinosus (and corresponding positions in Thiocapsa roseopersicina). Alanine mutants at these positions were found to be completely inactive, suggesting their direct involvement in the catalytic reaction. Our resulting mechanistic hypothesis is in analogy with the recently studied class of terpenoid cyclase enzymes containing a highly acidic aspartic residue in their active site. We propose that a similar aspartic acid residue, which is conserved through all putative CrtCs, is involved in initial protonation of the double bond in lycopene.BT/BiotechnologyApplied Science

A nano power CMOS tinnitus detector for a fully implantable closed-loop neurodevice

Analog signal processing offers advantages from a power consumption viewpoint. The real-time tinnitus detection method described in this paper detects tinnitus by comparing ECoG/EEG signal energies from different locations in the brain according to a tinnitus "signature". First, the proposed strategy selects appropriate ECoG/EEG bands per channel by means of band-pass filters. Next, their extracted energies are compared to their counterparts from a different (healthy) location. Tinnitus is detected only if higher theta and gamma energies associated with lower alpha energy, in comparison to corresponding signal energies from healthy brain region, are present. To verify the detector performance, a tinnitus CMOS detector circuit has been designed to be implemented in AMIS 0.35μm technology (I3T25) and has been verified by means of simulations in Cadence using RF spectre. The final circuit operates from a 1V supply and consumes only 60nA. The applicability of the detector is verified by means of circuit simulations with real neural waveforms and is able to successfully detect tinnitus. © 2011 IEEE

Enzyme-catalyzed cationic epoxide rearrangements in quinolone alkaloid biosynthesis

Epoxides are highly useful synthons and biosynthons in the construction of complex natural products during total synthesis and biosynthesis, respectively. Among enzyme-catalyzed epoxide transformations, a notably missing reaction, compared to the synthetic toolbox, is cationic rearrangement that takes place under strong acids. This is a challenging transformation for enzyme catalysis, as stabilization of the carbocation intermediate upon epoxide cleavage is required. Here, we discovered two Brønsted acid enzymes that can catalyze two unprecedented epoxide transformations in biology. PenF from the penigequinolone pathway catalyzes a cationic epoxide rearrangement under physiological conditions to generate a quaternary carbon center, while AsqO from the aspoquinolone pathway catalyzes a 3-exo-tet cyclization to forge a cyclopropane-tetrahydrofuran ring system. The discovery of these new epoxide-modifying enzymes further highlights the versatility of epoxides in complexity generation during natural product biosynthesis