How essential is the ‘essential’ active-site lysine in dihydrodipicolinate synthase?

Dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52), a validated antibiotic target, catalyses the first committed step in the lysine biosynthetic pathway: the condensation reaction between (S)-aspartate β-semialdehyde [(S)-ASA] and pyruvate via the formation of a Schiff base intermediate between pyruvate and the absolutely conserved active-site lysine. Escherichia coli DHDPS mutants K161A and K161R of the active-site lysine were characterised for the first time. Unexpectedly, the mutant enzymes were still catalytically active, albeit with a significant decrease in activity. The k_cat values for DHDPS-K161A and DHDPS-K161R were 0.06 ± 0.02 s⁻¹ and 0.16 ± 0.06 s⁻¹ respectively, compared to 45 ± 3 s⁻¹ for the wild-type enzyme. Remarkably, the K_M values for pyruvate increased by only 3-fold for DHDPS-K161A and DHDPS-K161R (0.45 ± 0.04 mM and 0.57 ± 0.06 mM, compared to 0.15 ± 0.01 mM for the wild-type DHDPS), while the K_M values for (S)-ASA remained the same for DHDPS-K161R (0.12 ± 0.01 mM) and increased by only 2-fold for DHDPS-K161A (0.23 ± 0.02 mM) and the K_i for lysine was unchanged. The X-ray crystal structures of DHDPS-K161A and DHDPS-K161R were solved at resolutions of 2.0 and 2.1 Å respectively and showed no changes in their secondary or tertiary structures when compared to the wild-type structure. The crystal structure of DHDPS-K161A with pyruvate bound at the active site was solved at a resolution of 2.3 Å and revealed a defined binding pocket for pyruvate that is thus not dependent upon lysine 161. Taken together with ITC and NMR data, it is concluded that although lysine 161 is important in the wild-type DHDPS-catalysed reaction, it is not absolutely essential for catalysis.     

Borane‐mediated asymmetric reduction of acetophenone by enantiopure aminonaphthols and aminoalcohols as catalytic source

Practical, cheap, and stereoselective synthetic methods were applied to the preparation of novel 1‐(aminoalkyl)naphthol and γ‐aminoalcohol tridentate ligands. The ligands obtained were conveniently applied with good results as catalytic sources in the borane‐mediated enantioselective reduction of acetophenone with borane dimethylsulfide. Conformational analysis through molecular modeling allows the rationalization of observed stereochemical outcomes. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

K_2CO_3/γ-Al_2O_3催化棕榈油和甲醇酯交换反应制备生物柴油

采用浸渍法制备K2CO3/γ-Al2O3负载型固体碱催化剂,用X线衍射(XRD)和热质量分析法(DSC-TGA)表征催化剂的物化性质,考察催化剂在棕榈油和甲醇酯交换制备生物柴油中的反应性能。结果表明:活性组分已成功负载到载体γ-Al2O3上,且在高温焙烧过程中K2CO3和γ-Al2O3之间产生了相互作用;在K2CO3负载量22.6%、醇油摩尔比12∶1、反应时间3h、催化剂质量分数3%、反应温度65℃的条件下,甲酯产率最高可达91.6%。     

Combining substrate dynamics, binding statistics, and energy barriers to rationalize regioselective hydroxylation of octane and lauric acid by CYP102A1 and mutants

Hydroxylations of octane and lauric acid by Cytochrome P450-BM3 (CYP102A1) wild-type and three active site mutants--F87A, L188Q/A74G, and F87V/L188Q/A74G--were rationalized using a combination of substrate orientation from docking, substrate binding statistics from molecular dynamics simulations, and barrier energies for hydrogen atom abstraction from quantum mechanical calculations. Wild-type BM3 typically hydroxylates medium- to long-chain fatty acids on subterminal (omega-1, omega-2, omega-3) but not the terminal (omega) positions. The known carboxylic anchoring site Y51/R47 for lauric acid, and hydrophobic interactions and steric exclusion, mainly by F87, for octane as well as lauric acid, play a role in the binding modes of the substrates. Electrostatic interactions between the protein and the substrate strongly modulate the substrate's regiodependent activation barriers. A combination of the binding statistics and the activation barriers of hydrogen-atom abstraction in the substrates is proposed to determine the product formation. Trends observed in experimental product formation for octane and lauric acid by wild-type BM3 and the three active site mutants were qualitatively explained. It is concluded that the combination of substrate binding statistics and hydrogen-atom abstraction barrier energies is a valuable tool to rationalize substrate binding and product formation and constitutes an important step toward prediction of product ratios.     

Accumulation of gold nanoparticles in Brassic juncea

Enzymatic digestion is proposed as a method for concentrating gold nanoparticles produced in plants. The mild conditions of digestion are used in order to avoid an increase in the gold particle size, which would occur with a high-temperature process, so that material suitable for catalysis may be produced. Gold nanoparticles of a 5-50-nm diameter, as revealed by transmission electron microscopy (TEM), at concentrations 760 and 1120 ppm Au, were produced within Brassica juncea grown on soil with 22-48 mg Au kg(-1). X-ray absorption near edge spectroscopy (XANES) reveals that the plant contained approximately equal quantities of Au in the metallic (Au0) and oxidized (Au+1) states. Enzymatic digestion dissolved 55-60 wt% of the plant matter. Due to the loss of the soluble gold fraction, no significant increase in the total concentration of gold in the samples was observed. However, it is likely that the concentration of the gold nanoparticles increased by a factor of two. To obtain a gold concentration suitable for catalytic reactions, around 95 wt% of the starting dry biomass would need to be solubilized or removed, which has not yet been achieved.     

Enzyme catalytic nitration of aromatic compounds

Nitroaromatic compounds are important intermediates in organic synthesis. The classic method used to synthesize them is chemical nitration, which involves the use of nitric acid diluted in water or acetic acid, both harmful to the environment. With the development of green chemistry, environmental friendly enzyme catalysis is increasingly employed in chemical processes. In this work, we adopted a non-aqueous horseradish peroxidase (HRP)/NaNO₂/H₂O₂ reaction system to study the structural characteristics of aromatic compounds potentially nitrated by enzyme catalysis, as well as the relationship between the charges on carbon atoms in benzene ring and the nitro product distribution. Investigation of various reaction parameters showed that mild reaction conditions (ambient temperature and neutral pH), plus appropriate use of H₂O₂ and NaNO₂ could prevent inactivation of HRP and polymerization of the substrates. Compared to aqueous–organic co-solvent reaction media, the aqueous–organic two-liquid phase system had great advantages in increasing the dissolved concentration of substrate and alleviating substrate inhibition. Analysis of the aromatic compounds’ structural characteristics indicated that substrates containing substituents of NH₂ or OH were readily catalyzed. Furthermore, analysis of the relationship between natural bond orbital (NBO) charges on carbon atoms in benzene ring, as calculated by the density functional method, and the nitro product distribution characteristics, demonstrated that the favored nitration sites were the ortho and para positions of substituents in benzene ring, similar to the selectivity of chemical nitration.     

Computational characterization of the chemical step in the GTP hydrolysis by Ras‐GAP for the wild‐type and G13V mutated Ras

The free energy profiles for the chemical reaction of the guanosine triphosphate hydrolysis GTP + H₂O → GDP + Pi by Ras‐GAP for the wild‐type and G13V mutated Ras were computed by using molecular dynamics protocols with the QM(ab initio)/MM potentials. The results are consistent with the recent measurements of reaction kinetics in Ras‐GAP showing about two‐order reduction of the rate constant upon G13V mutation in Ras: the computed activation barrier on the free energy profile is increased by 3 kcal/mol upon the G13V replacement. The major reason for a higher energy barrier is a shift of the “arginine finger” (R789 from GAP) from the favorable position in the active site. The results of simulations provide support for the mechanism of the reference reaction according to which the Q61 side chain directly participates in chemical transformations at the proton transfer stage. Proteins 2015; 83:1046–1053. © 2015 Wiley Periodicals, Inc.     

Proton reduction at surface of transition metal nanocatalysts

Catalytic activities of neutral and charged palladium (Pd) nanoparticles are compared for hydrogen reduction half-reaction. In this work the sequential H₂ dissociation from the surface of a Pd₁₃H₂₄ cluster is systematically studied by ab initio molecular dynamics (AIMD) at the density functional theory level. AIMD simulation is launched by preparing initial values of momenta of all nuclei in the model corresponding to a temperature range of 0–1700 K. AIMD simulation provides the trajectories of all the atoms in the cluster. A sequential H₂ desorption up to seven molecules is observed from the cluster surface due to thermal motion of nuclei. Modifications of total charge on the neutral Pd₁₃H₂₄ cluster model are found to affect surface H₂ desorption behaviour. A desorption rate of H₂ molecule on both neutral and charged Pd₁₃H₂₄ clusters is compared to the data of Pt₁₃H₂₄ cluster reported previously. The H₂ desorption energy on all the investigated clusters is also determined. The results reveal that Pd₁₃ cluster presents a higher catalytic activity than Pt₁₃ cluster.