Journal of Biological Chemistry最新文献

Unlike terrestrial environments, where humans reside, there is no sunlight in the deep sea. Instead, dim visible light from black-body radiation and bioluminescence illuminates hydrothermal vent areas in the deep sea. A deep-sea hydrothermal vent shrimp, Rimicaris hybisae, is thought to detect this dim light using its enlarged dorsal eye; however, the molecular basis of its photoreception remains unexplored. Here, we characterized the molecular properties of opsins, universal photoreceptive proteins in animals, found in R. hybisae. Transcriptomic analysis identified six opsins: three Gq-coupled opsins, one Opn3, one Opn5, and one peropsin. Functional analysis revealed that five of these opsins exhibited light-dependent G protein activity, whereas peropsin exhibited the ability to convert all-trans-retinal to 11-cis-retinal like photoisomerases. Notably, all the R. hybisae opsins, including Opn5, convergently show visible light sensitivity (around 457-517 nm), whereas most opsins categorized as Opn5 have been demonstrated to be UV sensitive. Mutational analysis revealed that the unique visible light sensitivity of R. hybisae Opn5 is achieved through the stabilization of a protonated Schiff base by a counterion residue at position 83 (Asp83), which differs from the position identified in other opsins. These findings suggest that the vent shrimp R. hybisae has adapted its photoreceptive devices to dim deep-sea hydrothermal light by selectively maintaining a repertoire of visible light-sensitive opsins, including the uniquely tuned Opn5.

Hydrogen (H) atoms account for about half the atoms in biomacromolecules and are essential for their biochemical properties such as enzymatic functions. Obtaining precise enzyme structures that include all the H atoms allows a deeper understanding of their structure-function relationships. Copper-containing nitrite reductases (CuNIRs) catalyze transformation of nitrite to nitric oxide, which has impacts on geochemical, agricultural, and medical health fields. Despite intense research efforts, the dynamics of H atoms during the enzymatic reaction of CuNIRs are unknown and hence the catalytic mechanism remains unclear. We performed neutron crystallography to shoot a single H-atom resolution picture of a CuNIR in complex with nitrite. We found that nitrite binds on the catalytic Cu center as nitrite (NO₂^-) and not as protonated HNO₂. Our X-ray data and quantum chemical calculation show that NO₂^- is in an electron-localized state that can facilitate N-O bond cleavage after receiving an electron. The catalytic residues, Asp^CAT and His^CAT, are deprotonated and protonated, respectively, suggesting that His^CAT is the point of departure of the proton transfer sequence. Quantum chemical calculations show that the neutron structure is consistent with the Cu(II) state and that the highly polarized state of the catalytic site is stabilized by the permittivity of solvent molecules filling a water channel. Subatomic resolution X-ray structures of the Asp^CAT-to-Asn mutants, which mimic the protonated state of Asp^CAT, were also determined to investigate the involvement of protonated Asp^CAT in the reaction. Our crystallographic data and quantum chemical calculations reveal in detail the first step of the CuNIR reaction.

Omega transaminases (ω-TAs) can mediate the chiral amination of several unnatural substrates without the requirement of an α-COOH group and are highly relevant in the production of several pharmaceutical intermediates of commercial interest. Development of better variants of ω-TAs is hence essential for the biotransformation of unnatural substrates. We studied the active site architecture of the wild-type ω-TAs, to engineer enzymes that enhance the biotransformation of (R)-phenylacetylcarbinol to (1R, 2S)-norephedrine. Two such ω-TAs (TA_5182 and TA_2799) from P. putida KT2440 strain were overexpressed and purified as recombinant proteins. Crystal structures of TA_5182 were solved in two conformations, revealing significant movements of two highly flexible loops in these different states. The TA_2799 structure was determined as a complex with the cofactor pyridoxal 5'-phosphate (PLP) covalently bound to the catalytic K286 as an internal aldimine. Enzyme assays indicated that TA_2799 required a four-fold higher cofactor concentration than TA_5182 to achieve satisfactory biotransformation of (R)-PAC. A key mutation of L322F in TA_2799 drastically reduced (∼8-fold) the cofactor dependency of the TA_2799_L322F mutant enzyme, and the mutant remained active for 96 h at 30 °C. The crystal structure of the mutant enzyme revealed a key asparagine residue that mediates a hydrogen bonding network at the dimeric interface of the enzyme and is absent in TA_5182. The TA_5182_G119N mutant also showed enhanced cofactor affinity. The results of our studies will help generate Pseudomonad ω-TAs and ω-TAs from other organisms with high efficiency for asymmetric synthesis, for further applications in large-scale biotransformation processes.

To gain insight into how ERG translocations cause prostate cancer, we performed single cell transcriptional profiling of an autochthonous mouse model at an early stage of disease initiation. Despite broad expression of ERG in all prostate epithelial cells, proliferation was enriched in a small, stem-like population with mixed-luminal basal identity (called intermediate cells). Through a series of lineage tracing and primary prostate tissue transplantation experiments, we find that tumor initiating activity resides in a subpopulation of basal cells that co-express the luminal genes Tmprss2 and Nkx3.1 (called Basal^Lum) but not in the larger population of classical Krt8+ luminal cells. Upon ERG activation, Basal^Lum cells give rise to the highly proliferative intermediate state, which subsequently transitions to the larger population of Krt8+ luminal cells characteristic of ERG-positive human cancers. Furthermore, this proliferative population is characterized by an ERG-specific chromatin state enriched for NFkB, AP-1, STAT and NFAT binding, with implications for TF cooperativity. The fact that the proliferative potential of ERG is enriched in a small stem-like population implicates the chromatin context of these cells as a critical variable for unmasking its oncogenic activity.