No abstract

Diaryl ketones are important building blocks for synthesizing pharmaceuticals and are generally regarded as “difficult-to-reduce” ketones due to the large steric hindrance of their two bulky aromatic side chains. Alcohol dehydrogenase from Kluyveromyces polyspora (KpADH) has been identified as a robust biocatalyst due to its high conversion of diaryl ketone substrate (4-chlorophenyl)­(pyridine-2-yl)­ketone (CPMK) with a moderate R-selectivity of 82% ee. To modulate the stereoselectivity of KpADH, a “polarity scanning” strategy was proposed, in which six key residues inside and at the entrance of the substrate binding pocket were identified. After iterative combinatorial mutagenesis, variants Mu-R2 and Mu-S5 with enhanced (99.2% ee, R) and inverted (97.8% ee, S) stereoselectivity were obtained. The crystal structures of KpADH and two mutants in complex with NADPH were resolved to elucidate the evolution of enantioselective inversion. Based on MD simulation, Mu-R2–CPMKProR and Mu-S5–CPMKProS were more favorable in the formation of prereaction states. Interestingly, a quadrilateral plane formed by α-carbons of four residues (N136, V161, C237, and G214) was identified at the entrance of the substrate binding pocket of Mu-S5; this plane acts as a “polar gate” for substrates. Due to the discrepancy in charge characteristics between chlorophenyl and pyridine substituents, the pro-S orientation of CPMK is defined when it passes through the “polar gate” in Mu-S5, whereas the similar plane in wild-type is blocked by several aromatic residues. Our result paves the way for engineering stereocomplementary ADH toward bulky diaryl ketones and provides structural insight into the mechanism of stereoselective inversion.

No abstract

No abstract

Density functional theory calculations (ωB97X-D) are reported for the reactions of methoxy, tert-butoxy, trichloroethoxy, and trifluoroethoxy radicals with a series of 26 C−H bonds in different environments characteristic of a variety of hydrocarbons and substituted derivatives. The variations in activation barriers are analyzed with modified Evans−Polanyi treatments to account for polarity and unsaturation effects. The treatments by Roberts and Steel and by Mayer have inspired the development of a simple treatment involving the thermodynamics of reactions, the difference between the reactant radical and product radical electronegativities, and the absence or presence of α-unsaturation. The three-parameter equation (ΔH ⧧ = 0.52ΔH rxn (1 − d) − 0.35Δχ AB 2 + 10.0, where d = 0.44 when there is α-unsaturation to the reacting C−H bond), correlates well with quantum mechanically computed barriers and shows the quantitative importance of the thermodynamics of reactions (dictated by the reactant and the product bond dissociation energies) and polar effects.

A D-carbamoylase NiHyuC from Nitratireductor indicus was identified with high catalytic activity toward Ncarbamoyl-D-tryptophan (3a). To further enhance its efficiency, both random mutagenesis and structure-guided evolution were performed. Variant M4 (D187N/A200N/S207A/R211G) showed a 43-fold increase in catalytic efficiency (k cat /K m = 1135.0 min −1 mM −1 ) and a 21-fold reduction in K m value (0.4 mM) compared with WT. Crystal structures of beneficial variants were resolved to clarify the evolutionary changes underlying improvements to catalytic efficiency. Structure alignment with WT indicated that loop 200−207 may play an important role in modulating access to the substrate entrance tunnel. Furthermore, MD simulations of WT−3a and M4−3a interactions illustrated that M4−3a has a better angle for nucleophilic attack and more readily enters a prereaction state. Additional hydrogen bonds and hydrophobic interactions were observed in prereaction states of M4−3a compared with that of WT−3a, consistent with its decreased K m value. In a hydantoinase process, the complete conversion of 160 mM Lindolylmethylhydantoin was achieved by M4 in a 0.5 L reaction, with D-tryptophan yield of 99.3% and productivity of 64.9 g L −1 d −1 . This study reveals a key loop at the substrate tunnel of D-carbamoylase and provides an effective strategy for engineering Dcarbamoylase and other carbon−nitrogen hydrolase family enzymes.

A novel 2-deoxyribose-5-phosphate aldolase (LbDERA) was identified from Lactobacillus brevis, with high activity, excellent thermostability and high tolerance against aldehyde substrates. The half-lives of LbDERA incubated in 300 mM acetaldehyde and chloroacetaldehyde were 37.3 and 198 min, respectively, which are 2-and 7-fold higher than those of EcDERA from Escherichia coli. The crystal structure of LbDERA determined at 1.95 Å resolution revealed a stable quaternary structure which might account for its excellent aldehyde tolerance. A single mutation, E78K, was introduced to LbDERA through a consensus sequence approach, resulting in significant improvements of both thermostability and aldehyde tolerance. According to the crystal structure of LbDERA E78K , two additional hydrogen bonds and one salt bridge were introduced compared with wild-type LbDERA. As a result of its high substrate tolerance, LbDERA E78K could efficiently catalyze a sequential aldol condensation with 0.7 M chloroacetaldehyde and 1.4 M acetaldehyde, affording a key chiral precursor of statins, IJ3R,5S)-6-chloro-2,4,6-trideoxyhexapyranoside, with an unprecedented space-time yield of 792.5 g L −1 d −1 and only 2.5 g L −1 of catalyst loading.

BackgroundEscherichia coli has been explored as a platform host strain for biofuels production such as butanol. However, the severe toxicity of butanol is considered to be one major limitation for butanol production from E. coli. The goal of this study is therefore to construct butanol-tolerant E. coli strains and clarify the tolerance mechanisms.ResultsA recombinant E. coli strain harboring σ70 mutation capable of tolerating 2 % (v/v) butanol was isolated by the global transcription machinery engineering (gTME) approach. DNA microarrays were employed to assess the transcriptome profile of butanol-tolerant strain B8. Compared with the wild-type strain, 329 differentially expressed genes (197 up-regulated and 132 down-regulated) (p < 0.05; FC ≥ 2) were identified. These genes are involved in carbohydrate metabolism, energy metabolism, two-component signal transduction system, oxidative stress response, lipid and cell envelope biogenesis and efflux pump.ConclusionsSeveral membrane-related proteins were proved to be involved in butanol tolerance of E. coli. Two down-regulated genes, yibT and yghW, were identified to be capable of affecting butanol tolerance by regulating membrane fatty acid composition. Another down-regulated gene ybjC encodes a predicted inner membrane protein. In addition, a number of up-regulated genes, such as gcl and glcF, contribute to supplement metabolic intermediates for glyoxylate and TCA cycles to enhance energy supply. Our results could serve as a practical strategy for the construction of platform E. coli strains as biofuel producer.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0527-9) contains supplementary material, which is available to authorized users.