A Novel Enzymatic System against Oxidative Stress in the Thermophilic Hydrogen-Oxidizing Bacterium Hydrogenobacter thermophilus

Rubrerythrin (Rbr) is a non-heme iron protein composed of two distinctive domains and functions as a peroxidase in anaerobic organisms. A novel Rbr-like protein, ferriperoxin (Fpx), was identified in Hydrogenobacter thermophilus and was found not to possess the rubredoxin-like domain that is present in typical Rbrs. Although this protein is widely distributed among aerobic organisms, its function remains unknown. In this study, Fpx exhibited ferredoxin:NADPH oxidoreductase (FNR)-dependent peroxidase activity and reduced both hydrogen peroxide (H2O2) and organic hydroperoxide in the presence of NADPH and FNR as electron donors. The calculated Km and Vmax values of Fpx for organic hydroperoxides were comparable to that for H2O2, demonstrating a multiple reactivity of Fpx towards hydroperoxides. An fpx gene disruptant was unable to grow under aerobic conditions, whereas its growth profiles were comparable to those of the wild-type strain under anaerobic and microaerobic conditions, clearly indicating the indispensability of Fpx as an antioxidant of H. thermophilus in aerobic environments. Structural analysis suggested that domain-swapping occurs in Fpx, and this domain-swapped structure is well conserved among thermophiles, implying the importance of structural stability of domain-swapped conformation for thermal environments. In addition, Fpx was located on a deep branch of the phylogenetic tree of Rbr and Rbr-like proteins. This finding, taken together with the wide distribution of Fpx among Bacteria and Archaea, suggests that Fpx is an ancestral type of Rbr homolog that functions as an essential antioxidant and may be part of an ancestral peroxide-detoxification system

Cysteine Nucleophiles in Glycosidase Catalysis : Application of a Covalent β-L-Arabinofuranosidase Inhibitor

The recent discovery of zinc-dependent retaining glycoside hydrolases (GHs), with active sites built around a Zn(Cys)(3)(Glu) coordination complex, has presented unresolved mechanistic questions. In particular, the proposed mechanism, depending on a Zn-coordinated cysteine nucleophile and passing through a thioglycosyl enzyme intermediate, remains controversial. This is primarily due to the expected stability of the intermediate C-S bond. To facilitate the study of this atypical mechanism, we report the synthesis of a cyclophellitol-derived beta-l-arabinofuranosidase inhibitor, hypothesised to react with the catalytic nucleophile to form a non-hydrolysable adduct analogous to the mechanistic covalent intermediate. This beta-l-arabinofuranosidase inhibitor reacts exclusively with the proposed cysteine thiol catalytic nucleophiles of representatives of GH families 127 and 146. X-ray crystal structures determined for the resulting adducts enable MD and QM/MM simulations, which provide insight into the mechanism of thioglycosyl enzyme intermediate breakdown. Leveraging the unique chemistry of cyclophellitol derivatives, the structures and simulations presented here support the assignment of a zinc-coordinated cysteine as the catalytic nucleophile and illuminate the finely tuned energetics of this remarkable metalloenzyme clan.Medical BiochemistryBio-organic Synthesi

The lactose operon from Lactobacillus casei is involved in the transport and metabolism of the human milk oligosaccharide core-2 N-acetyllactosamine

The lactose operon (lacTEGF) from Lactobacillus casei strain BL23 has been previously studied. The lacT gene codes for a transcriptional antiterminator, lacE and lacF for the lactose-specific phosphoenolpyruvate: phosphotransferase system (PTSLac) EIICB and EIIA domains, respectively, and lacG for the phospho-β-galactosidase. In this work, we have shown that L. casei is able to metabolize N-acetyllactosamine (LacNAc), a disaccharide present at human milk and intestinal mucosa. The mutant strains BL153 (lacE) and BL155 (lacF) were defective in LacNAc utilization, indicating that the EIICB and EIIA of the PTSLac are involved in the uptake of LacNAc in addition to lactose. Inactivation of lacG abolishes the growth of L. casei in both disaccharides and analysis of LacG activity showed a high selectivity toward phosphorylated compounds, suggesting that LacG is necessary for the hydrolysis of the intracellular phosphorylated lactose and LacNAc. L. casei (lacAB) strain deficient in galactose-6P isomerase showed a growth rate in lactose (0.0293 ± 0.0014 h-1) and in LacNAc (0.0307 ± 0.0009 h-1) significantly lower than the wild-type (0.1010 ± 0.0006 h-1 and 0.0522 ± 0.0005 h-1, respectively), indicating that their galactose moiety is catabolized through the tagatose-6P pathway. Transcriptional analysis showed induction levels of the lac genes ranged from 130 to 320-fold in LacNAc and from 100 to 200-fold in lactose, compared to cells growing in glucose

Probing the Mutational Interplay between Primary and Promiscuous Protein Functions: A Computational-Experimental Approach

Protein promiscuity is of considerable interest due its role in adaptive metabolic plasticity, its fundamental connection with molecular evolution and also because of its biotechnological applications. Current views on the relation between primary and promiscuous protein activities stem largely from laboratory evolution experiments aimed at increasing promiscuous activity levels. Here, on the other hand, we attempt to assess the main features of the simultaneous modulation of the primary and promiscuous functions during the course of natural evolution. The computational/experimental approach we propose for this task involves the following steps: a function-targeted, statistical coupling analysis of evolutionary data is used to determine a set of positions likely linked to the recruitment of a promiscuous activity for a new function; a combinatorial library of mutations on this set of positions is prepared and screened for both, the primary and the promiscuous activities; a partial-least-squares reconstruction of the full combinatorial space is carried out; finally, an approximation to the Pareto set of variants with optimal primary/promiscuous activities is derived. Application of the approach to the emergence of folding catalysis in thioredoxin scaffolds reveals an unanticipated scenario: diverse patterns of primary/promiscuous activity modulation are possible, including a moderate (but likely significant in a biological context) simultaneous enhancement of both activities. We show that this scenario can be most simply explained on the basis of the conformational diversity hypothesis, although alternative interpretations cannot be ruled out. Overall, the results reported may help clarify the mechanisms of the evolution of new functions. From a different viewpoint, the partial-least-squares-reconstruction/Pareto-set-prediction approach we have introduced provides the computational basis for an efficient directed-evolution protocol aimed at the simultaneous enhancement of several protein features and should therefore open new possibilities in the engineering of multi-functional enzymes

Two distinct catalytic pathways for GH43 xylanolytic enzymes unveiled by X-ray and QM/MM simulations

Xylanolytic enzymes from glycoside hydrolase family 43 (GH43) are involved in the breakdown of hemicellulose, the second most abundant carbohydrate in plants. Here, we kinetically and mechanistically describe the non-reducing-end xylose-releasing exo-oligoxylanase activity and report the crystal structure of a native GH43 Michaelis complex with its substrate prior to hydrolysis. Two distinct calcium-stabilized conformations of the active site xylosyl unit are found, suggesting two alternative catalytic routes. These results are confirmed by QM/MM simulations that unveil the complete hydrolysis mechanism and identify two possible reaction pathways, involving different transition state conformations for the cleavage of xylooligosaccharides. Such catalytic conformational promiscuity in glycosidases is related to the open architecture of the active site and thus might be extended to other exo-acting enzymes. These findings expand the current general model of catalytic mechanism of glycosidases, a main reaction in nature, and impact on our understanding about their interaction with substrates and inhibitors

Surface adhesion reduction in silicon microstructures using femtosecond laser pulses

A reduction of the adhesion between polysilicon surface-micromachined structures and its silicon substrate using ultrashort pulse laser irradiation has been demonstrated. Polysilicon cantilevers, which adhered to the silicon substrate after final rinse and dry, were freed after irradiation by a 800 nm wavelength laser with pulse duration of 150 fs (full width at half-maximum) and fluences up to 40 mJ/cm2. Increasing the pulse widths to 2.7 ps resulted in significantly fewer freed cantilevers indicating that the process depends heavily on the presence of high-temperature carriers in the silicon. Adhesion reduction has been observed from exposure to a single pulse which results in minimal lattice temperature increase. © 1996 American Institute of Physics.link_to_subscribed_fulltex