Primary alcohol oxidation by aryl‐alcohol oxidase (AAO), a flavoenzyme providing H2O2 to ligninolytic peroxidases, is produced by concerted proton and hydride transfers, as shown by substrate and solvent kinetic isotope effects (KIEs). Interestingly, when the reaction was investigated with synthesized (R)‐ and (S)‐α‐deuterated p‐methoxybenzyl alcohol, a primary KIE (≈6) was observed only for the R enantiomer, revealing that the hydride transfer is highly stereoselective. Docking of p‐methoxybenzyl alcohol at the buried crystal active site, together with QM/MM calculations, showed that this stereoselectivity is due to the position of the hydride‐ and proton‐receiving atoms (flavin N5 and His502 Nε, respectively) relative to the alcohol Cα‐substituents, and to the concerted nature of transfer (the pro‐S orientation corresponding to a 6 kcal mol?1 penalty with respect to the pro‐R orientation). The role of His502 is supported by the lower activity (by three orders of magnitude) of the H502A variant. The above stereoselectivity was also observed, although activities were much lower, in AAO reactions with secondary aryl alcohols (over 98 % excess of the R enantiomer after treatment of racemic 1‐(p‐methoxyphenyl)ethanol, as shown by chiral HPLC) and especially with use of the F501A variant. This variant has an enlarged active site that allow better accommodation of the α‐substituents, resulting in higher stereoselectivity (S/R ratios) than is seen with AAO. High enantioselectivity in a member of the GMC oxidoreductase superfamily is reported for the first time, and shows the potential for engineering of AAO for deracemization purposes. 相似文献
An exo-β-xylosidase mutant with glycosynthase activity was created to aid in the synthesis of xylanase substrates and inhibitors. Simple monosaccharides were easily elaborated into di-, tri- and tetrasaccharides by using this enzyme. Some products proved to be surprisingly potent inhibitors of xylanases from glycoside hydrolase families 10 and 11. 相似文献
MLL3, also known as KMT2C, is a lysine mono-methyltransferase in charge of the writing of an epigenetic mark on lysine 4 from histone 3. The catalytic site of MLL3 is composed of four tyrosines, namely, Y44, Y69, Y128, and Y130. Tyrosine residues are highly conserved among lysine methyltransferases’ catalytic sites, although their complete function is still unclear. The exploration of how modifications on these residues from the enzymatic machinery impact the enzymatic activity of MLL3 could shed light transversally into the inner functioning of enzymes with similar characteristics. Through the use of QMMM calculations, we focus on the effect of the mutation of each tyrosine from the catalytic site on the enzymatic activity and the product specificity in the current study. While we found that the mutations of Y44 and Y128 by phenylalanine inactivated the enzyme, the mutation of Y128 by alanine reactivated the enzymatic activity of MLL3. Moreover, according to our models, the Y128A mutant was even found to be capable of di- and tri-methylate lysine 4 from histone 3, what would represent a gain of function mutation, and could be responsible for the development of diseases. Finally, we were able to establish the inactivation mechanism, which involved the use of Y130 as a water occlusion structure, whose conformation, once perturbed by its mutation or Y128 mutant, allows the access of water molecules that sequester the electron pair from lysine 4 avoiding its methylation process and, thus, increasing the barrier height. 相似文献
The use of gas-phase iodine and carbon dioxide as transport agents in the tantalum/carbon/tantalum carbide combustion synthesis system has been examined to determine the effects of transport agents on product composition and microstructure. Two tantalum reactant particle sizes, a range of transport agent concentrations, and total pressures were studied. The effects of the combustion conditions on product morphology and composition were evaluated using scanning electron microscopy, nitrogen adsorption (specific surface area), and X-ray diffraction analyses. The results of the investigation indicate that the presence of the iodine vapor and carbon dioxide significantly enhances the combustion synthesis process, leading to higher conversion efficiencies and influencing product microstructure. The results are discussed in the context of gas-phase and solid-phase transport models. 相似文献
A general method for the catalytic asymmetric synthesis of 4‐alkyl‐4H‐chromenes was developed. With readily available β‐alkyl‐substituted enones and 2‐hydroxylated arylboronic acids, a rhodium‐catalysed asymmetric conjugate addition/intramolecular hemi‐acetalization/acid‐promoted dehydration sequence leads to the formation of 4‐alkyl‐4H‐chromenes in up to 99% yield and with up to >99% ee. The current study remedies the methodological deficiency in asymmetric synthesis of 4‐alkyl‐4H‐chromenes.
LTA4H is a bifunctional zinc metalloenzyme that converts leukotriene A4 (LTA4) into leukotriene B4 (LTB4), one of the most potent chemotactic agents involved in acute and chronic inflammatory diseases. In this reaction, LTA4H acts as an epoxide hydrolase with a unique and fascinating mechanism, which includes the stereoselective attachment of one water molecule to the carbon backbone of LTA4 several methylene units away from the epoxide moiety. By combining Molecular Dynamics simulations and Quantum Mechanics/Molecular Mechanics calculations, we obtained a very detailed molecular picture of the different consecutive steps of that mechanism. By means of a rather unusual 1,7-nucleophilic substitution through a clear SN1 mechanism, the epoxide opens and the triene moiety of the substrate twists in such a way that the bond C6-C7 adopts its cis (Z) configuration, thus exposing the R face of C12 to the addition of a water molecule hydrogen-bonded to ASP375. Thus, the two stereochemical features that are required for the bioactivity of LTB4 appear to be closely related. The noncovalent π-π stacking interactions between the triene moiety and two tyrosines (TYR267 and, especially, TYR378) that wrap the triene system along the whole reaction explain the preference for the cis configuration inside LTA4H. 相似文献
