In silico structure–function analysis of E. cloacae nitroreductase

Reduction, catalyzed by the bacterial nitroreductases, is the quintessential first step in the biodegradation of a variety of nitroaromatic compounds from contaminated waters and soil. The Enterobacter cloacae nitroreductase (EcNR) enzyme is considered as a prospective biotechnological tool for bioremediation of hazardous nitroaromatic compounds. Using diverse computational methods, we obtain insights into the structural basis of activity and mechanism of its function. We have performed molecular dynamics simulation of EcNR in three different states (free EcNR in oxidized form, fully reduced EcNR with benzoate inhibitor and fully reduced EcNR with nitrobenzene) in explicit solvent and with full electrostatics. Principal Component Analysis (PCA) of the variance‐covariance matrix showed that the complexed nitroreductase becomes more flexible overall upon complexation, particularly helix H6, in the vicinity of the binding site. A multiple sequence alignment was also constructed in order to examine positional constraints on substitution in EcNR. Five regions which are highly conserved within the flavin mononucleotide (FMN) binding site were identified. Obtained results and their implications for EcNR functioning are discussed, and new plausible mechanism has been proposed. Proteins 2012; © 2012 Wiley Periodicals, Inc.     

Interaction of J-protein co-chaperone Jac1 with Fe-S scaffold Isu is indispensable in vivo and conserved in evolution

The ubiquitous mitochondrial J-protein Jac1, called HscB in Escherichia coli, and its partner Hsp70 play a critical role in the transfer of Fe-S clusters from the scaffold protein Isu to recipient proteins. Biochemical results from eukaryotic and prokaryotic systems indicate that formation of the Jac1-Isu complex is important for both targeting of the Isu for Hsp70 binding and stimulation of Hsp70's ATPase activity. However, in apparent contradiction, we previously reported that an 8-fold decrease in Jac1's affinity for Isu1 is well tolerated in vivo, raising the question as to whether the Jac1:Isu interaction actually plays an important biological role. Here, we report the determination of the structure of Jac1 from Saccharomyces cerevisiae. Taking advantage of this information and recently published data from the homologous bacterial system, we determined that a total of eight surface-exposed residues play a role in Isu binding, as assessed by a set of biochemical assays. A variant having alanines substituted for these eight residues was unable to support growth of a jac1-Δ strain. However, replacement of three residues caused partial loss of function, resulting in a significant decrease in the Jac1:Isu1 interaction, a slow growth phenotype, and a reduction in the activity of Fe-S cluster-containing enzymes. Thus, we conclude that the Jac1:Isu1 interaction plays an indispensable role in the essential process of mitochondrial Fe-S cluster biogenesis.     

Plastoquinol is more active than α-tocopherol in singlet oxygen scavenging during high light stress of Chlamydomonas reinhardtii

In the present study, we have performed comparative analysis of different prenyllipids in Chlamydomonas reinhardtii cultures during high light stress under variety of conditions (presence of inhibitors, an uncoupler, heavy water). The obtained results indicate that plastoquinol is more active than α-tocopherol in scavenging of singlet oxygen generated in photosystem II. Besides plastoquinol, also its oxidized form, plastoquinone shows antioxidant action during the stress conditions, resulting in formation of plastoquinone-C, whose level can be regarded as an indicator of singlet oxygen oxidative stress in vivo. The pronounced stimulation of α-tocopherol consumption and α-tocopherolquinone formation by an uncoupler, FCCP, together with the results of additional model system studies, led to the suggestion that α-tocopherol can be recycled in thylakoid membranes under high light conditions from 8a-hydroperoxy-α-tocopherone, the primary oxidation product of α-tocopherol by singlet oxygen.     

Singlet oxygen and non-photochemical quenching contribute to oxidation of the plastoquinone-pool under high light stress in Arabidopsis

The redox state of plastoquinone-pool in chloroplasts is crucial for driving many responses to variable environment, from short-term effects to those at the gene expression level. In the present studies, we showed for the first time that the plastoquinone-pool undergoes relatively fast oxidation during high light stress of low light-grown Arabidopsis plants. This oxidation was not caused by photoinhibition of photosystem II, but mainly by singlet oxygen generated in photosystem II and non-photochemical quenching in light harvesting complex antenna of the photosystem, as revealed in experiments with a singlet oxygen scavenger and with Arabidopsis npq4 mutant. The latter mechanism suppresses the influx of electrons to the plastoquinone-pool preventing its excessive reduction. The obtained results are of crucial importance in light of the function of the redox state of the plastoquinone-pool in triggering many high light-stimulated physiological responses of plants.     

Reversible lysine acetylation regulates activity of human glycine N-acyltransferase-like 2 (hGLYATL2): implications for production of glycine-conjugated signaling molecules

Lysine acetylation is a major post-translational modification of proteins and regulates many physiological processes such as metabolism, cell migration, aging, and inflammation. Proteomic studies have identified numerous lysine-acetylated proteins in human and mouse models (Kim, S. C., Sprung, R., Chen, Y., Xu, Y., Ball, H., Pei, J., Cheng, T., Kho, Y., Xiao, H., Xiao, L., Grishin, N. V., White, M., Yang, X. J., and Zhao, Y. (2006) Mol. Cell 23, 607-618). One family of proteins identified in this study was the murine glycine N-acyltransferase (GLYAT) enzymes, which are acetylated on lysine 19. Lysine 19 is a conserved residue in human glycine N-acyltransferase-like 2 (hGLYATL2) and in several other species, showing that this residue may be important for enzyme function. Mutation of lysine 19 in recombinant hGLYATL2 to glutamine (K19Q) and arginine (K19R) resulted in a 50-80% lower production of N-oleoyl glycine and N-arachidonoylglycine, indicating that lysine 19 is important for enzyme function. LC/MS/MS confirmed that Lys-19 is not acetylated in wild-type hGLYATL2, indicating that Lys-19 requires to be deacetylated for full activity. The hGLYATL2 enzyme conjugates medium- and long-chain saturated and unsaturated acyl-CoA esters to glycine, resulting in the production of N-oleoyl glycine and also N-arachidonoyl glycine. N-Oleoyl glycine and N-arachidonoyl glycine are structurally and functionally related to endocannabinoids and have been identified as signaling molecules that regulate functions like the perception of pain and body temperature and also have anti-inflammatory properties. In conclusion, acetylation of lysine(s) in hGLYATL2 regulates the enzyme activity, thus linking post-translational modification of proteins with the production of biological signaling molecules, the N-acyl glycines.     

Plant genome modification by homologous recombination

The mechanisms and frequencies of various types of homologous recombination (HR) have been studied in plants for several years. However, the application of techniques involving HR for precise genome modification is still not routine. The low frequency of HR remains the major obstacle but recent progress in gene targeting in Arabidopsis and rice, as well as accumulating knowledge on the regulation of recombination levels, is an encouraging sign of the further development of HR-based approaches for genome engineering in plants.