Structural coordination between active sites of a CRISPR reverse transcriptase-integrase complex.

CRISPR-Cas systems provide adaptive immunity in bacteria and archaea, beginning with integration of foreign sequences into the host CRISPR genomic locus and followed by transcription and maturation of CRISPR RNAs (crRNAs). In some CRISPR systems, a reverse transcriptase (RT) fusion to the Cas1 integrase and Cas6 maturase creates a single protein that enables concerted sequence integration and crRNA production. To elucidate how the RT-integrase organizes distinct enzymatic activities, we present the cryo-EM structure of a Cas6-RT-Cas1-Cas2 CRISPR integrase complex. The structure reveals a heterohexamer in which the RT directly contacts the integrase and maturase domains, suggesting functional coordination between all three active sites. Together with biochemical experiments, our data support a model of sequential enzymatic activities that enable CRISPR sequence acquisition from RNA and DNA substrates. These findings highlight an expanded capacity of some CRISPR systems to acquire diverse sequences that direct CRISPR-mediated interference.

Stability-Controllable Self-Immobilization of Carbonic Anhydrase Fused with a Silica-Binding Tag onto Diatom Biosilica for Enzymatic CO2 Capture and Utilization.

Exploiting carbonic anhydrase (CA), an enzyme that catalyzes the hydration of CO2, is a powerful route for eco-friendly and cost-effective carbon capture and utilization. For successful industrial applications, the stability and reusability of CA should be improved, which necessitates enzyme immobilization. Herein, the ribosomal protein L2 (Si-tag) from Escherichia coli was utilized for the immobilization of CA onto diatom biosilica, a promising renewable support material. The Si-tag was redesigned (L2NC) and genetically fused to CA from the marine bacterium Hydrogenovibrio marinus (hmCA). One-step self-immobilization of hmCA-L2NC onto diatom biosilica by simple mixing was successfully achieved via Si-tag-mediated strong binding, showing multilayer adsorption with a maximal loading of 1.4 wt %. The immobilized enzyme showed high reusability and no enzyme leakage even under high temperature conditions. The activity of hmCA-L2NC was inversely proportional to the enzyme loading, while the stability was directly proportional to the enzyme loading. This discovered activity-stability trade-off phenomenon could be attributed to macromolecular crowding on the highly dense surface of the enzyme-immobilized biosilica. Collectively, our system not only facilitates the stability-controllable self-immobilization of enzyme via Si-tag on a diatom biosilica support for the robust, facile, and green construction of stable biocatalysts, but is also a unique model for studying the macromolecular crowding effect on surface-immobilized enzymes.

MxaJ structure reveals a periplasmic binding protein-like architecture with unique secondary structural elements.

MxaJ is a component of type II methanol dehydrogenase (MDH) that mediates electron transfer during methanol oxidation in methanotrophic bacteria. However, little is known about how MxaJ structurally cooperates with MDH and Cytochrome cL . Here, we report for the first time the crystal structure of MxaJ. MxaJ consists of eight α-helices and six ß-strands, and resembles the "bi-lobate" folding architecture found in periplasmic binding proteins. Distinctive features of MxaJ include prominent loops and a ß-strand around the hinge region supporting the ligand-binding cavity, which might provide a more favorable framework for interacting with proteins rather than small molecules. Proteins 2017; 85:1379-1386. © 2017 Wiley Periodicals, Inc.

Redox-dependent structural transformations of the [4Fe-3S] proximal cluster in O2-tolerant membrane-bound [NiFe]-hydrogenase: a DFT study.

Broken-symmetry density functional theory (BS-DFT) has been used to address the redox-dependent structural changes of the proximal [4Fe-3S] cluster, implicated in the O2-tolerance of membrane-bound [NiFe]-hydrogenase (MBH). The recently determined structures of the [4Fe-3S] cluster together with its protein ligands were studied at the reduced [4Fe-3S](3+), oxidized [4Fe-3S](4+), and superoxidized [4Fe-3S](5+) levels in context of their relative energies and protonation states. The observed proximal cluster conformational switch, concomitant with the proton transfer from the cysteine Cys20 backbone amide to the nearby glutamate Glu76 carboxylate, is found to be a single-step process requiring ~12-17 kcal/mol activation energy at the superoxidized [4Fe-3S](5+) level. At the more reduced [4Fe-3S](4+/3+) oxidation levels, this rearrangement has at least 5 kcal/mol higher activation barriers and prohibitively unfavorable product energies. The reverse transformation of the proximal cluster is a fast unidirectional process with ~8 kcal/mol activation energy, triggered by one-electron reduction of the superoxidized species. A previously discussed ambiguity of the Glu76 carboxylate and 'special' Fe4 iron positions in the superoxidized cluster is now rationalized as a superposition of two local minima, where Glu76-Fe4 coordination is either present or absent. The calculated 12.3-17.9 MHz (14)N hyperfine coupling (HFC) for the Fe4-bound Cys20 backbone nitrogen is in good agreement with the large 13.0/14.6 MHz (14)N couplings from the latest HYSCORE/ENDOR studies.

Tryptophan-47 in the active site of Methylophaga sp. strain SK1 flavin-monooxygenase is important for hydride transfer.

Flavin-dependent monooxygenase (FMO) from Methylophaga sp. strain SK1 catalyzes the NADPH- and oxygen-dependent hydroxylation of a number of xenobiotics. Reduction of the flavin cofactor by NADPH is required for activation of molecular oxygen. The role of a conserved tryptophan at position 47 was probed by site-directed mutagenesis. FMOW47A resulted in an insoluble inactive protein; in contrast, FMOW47F was soluble and active. The spectrum of the flavin in the mutant enzyme was redshifted, indicating a change in the flavin environment. The kcat values for NADPH, trimethylamine, and methimazole, decreased 5-8-fold. Primary kinetic isotope effect values were higher, indicating that hydride transfer is more rate-limiting in the mutant enzyme. This is supported by a decrease in the rate constant for flavin reduction and in the solvent kinetic isotope effect values. Results from molecular dynamics simulations show reduced flexibility in active site residues and, in particular, the nicotinamide moiety of NADP+ in FMOW47F. This was supported by thermal denaturation experiments. Together, the data suggests that W47 plays a role in maintaining the overall protein flexibility that is required for conformational changes important in hydride transfer.

Production of novel antioxidative prenyl naphthalen-ols by combinational bioconversion with dioxygenase PhnA1A2A3A4 and prenyltransferase NphB or SCO7190.

We performed combinational bioconversion of substituted naphthalenes with PhnA1A2A3A4 (an aromatic dihydroxylating dioxygenase from marine bacterium Cycloclasticus sp. strain A5) and prenyltransferase NphB (geranyltransferase from Streptomyces sp. strain CL190) or SCO7190 (dimethylallyltransferase from Streptomyces coelicolor A3(2)) to produce prenyl naphthalen-ols. Using 2-methylnaphthalene, 1-methoxynaphthalene, and 1-ethoxynaphthalene as the starting substrates, 10 novel prenyl naphthalen-ols were produced by combinational bioconversion. These novel prenyl naphthalen-ols each showed potent antioxidative activity against a rat brain homogenate model. 2-(2,3-Dihydroxyphenyl)-5,7-dihydroxy-chromen-4-one (2',3'-dihydroxychrysin) generated with another aromatic dihydroxylating dioxygenase and subsequent dehydrogenase was also geranylated at the C-5'-carbon by the action of NphB.

Batch and continuous biooxidation of sulphide by Thiomicrospira sp. CVO: reaction kinetics and stoichiometry.

Aqueous phase biooxidation of sulphide by the novel sulphide-oxidizing bacterium Thiomicrospira sp. CVO was studied in batch and continuous systems. CVO was able to oxidize sulphide at concentrations as high as 19 mM. Sulphide biooxidation occurred in two distinct phases, one resulting in the formation of sulphur and possibly other dissolved sulphur compounds rather than sulphate, followed by sulphate formation. The specific growth rate of CVO in the first and second phases were 0.17-0.27 and 0.04-0.05 h(-1), respectively. Nitrite accumulated in the culture during the first phase and was consumed during the second phase. The composition of end-products was influenced by the ratio of sulphide to nitrate initial concentrations. At a ratio of 0.28, sulphate represented 93% of the reaction products, while with a ratio of 1.6 the conversion of sulphide to sulphate was only 9.3%. In the continuous bioreactor, complete removal of sulphide was observed at sulphide volumetric loading rates as high as 1.6mM/h (residence time of 10h). Overall sulphide removal efficiency decreased continuously upon further increases in volumetric loading rate. However, the volumetric removal rate increased until a maximum value of 2.4mM/h was obtained at a loading rate of 3.2mM/h. The corresponding sulphide conversion and residence time were 76% and 5.6h, respectively. As expected from the high ratio of sulphide to nitrate loading rates (1.7-1.9 mM/h), no sulphate was formed in the continuous reactor. Using the experimental data the value of maximum specific growth rate, saturation constant, decay coefficient, maintenance coefficient and yield were determined to be 0.36 h(-1), 1.99 mM sulphide, 0.0014 h(-1), 0.078 mmol sulphide/mg ATPh and 0.018 mg ATP/mmol sulphide, respectively.