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

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

Ultrashort-pulse laser heating of silicon to reduce microstructure adhesion

A technique to remove moisture from microelectronic devices and improve device yield in microelectromechanical systems by reducing microstructure surface adhesion is proposed. Ultrashort-pulse laser radiation is used to create excited carriers in, and consequently desorb water from, silicon microstructures. A theoretical model for ultrashort-pulse laser heating of silicon is presented. Calculated carrier temperatures show significant increases at short time scales, while the lattice temperatures remain almost constant, indicating the possibility for water desorption without significant device heating. A preliminary experiment confirming the feasibility of using the technique to decrease microstructure adhesion is discussed. Copyright © 1996 Elsevier Science Ltd.link_to_subscribed_fulltex

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