Diterpene synthase VenA is responsible for assembling venezuelaene A with a unique 5-5-6-7 tetracyclic skeleton from geranylgeranyl pyrophosphate. VenA also demonstrates substrate promiscuity by ...accepting geranyl pyrophosphate and farnesyl pyrophosphate as alternative substrates. Herein, we report the crystal structures of VenA in both apo form and holo form in complex with a trinuclear magnesium cluster and pyrophosphate group. Functional and structural investigations on the atypical
DSFVSD
motif of VenA, versus the canonical Asp-rich motif of DDXX(X)D/E, reveal that the absent second Asp of canonical motif is functionally replaced by Ser116 and Gln83, together with bioinformatics analysis identifying a hidden subclass of type I microbial terpene synthases. Further structural analysis, multiscale computational simulations, and structure-directed mutagenesis provide significant mechanistic insights into the substrate selectivity and catalytic promiscuity of VenA. Finally, VenA is semi-rationally engineered into a sesterterpene synthase to recognize the larger substrate geranylfarnesyl pyrophosphate.
Dimethylsulfoniopropionate (DMSP) is an abundant and ubiquitous organosulfur molecule in marine environments with important roles in global sulfur and nutrient cycling. Diverse DMSP lyases in some ...algae, bacteria, and fungi cleave DMSP to yield gaseous dimethyl sulfide (DMS), an infochemical with important roles in atmospheric chemistry. Here, we identified a novel ATP-dependent DMSP lyase, DddX. DddX belongs to the acyl-CoA synthetase superfamily and is distinct from the eight other known DMSP lyases. DddX catalyses the conversion of DMSP to DMS via a two-step reaction: the ligation of DMSP with CoA to form the intermediate DMSP-CoA, which is then cleaved to DMS and acryloyl-CoA. The novel catalytic mechanism was elucidated by structural and biochemical analyses. DddX is found in several Alphaproteobacteria, Gammaproteobacteria, and Firmicutes, suggesting that this new DMSP lyase may play an overlooked role in DMSP/DMS cycles.
The collagenases of Vibrio species, many of which are pathogens, have been regarded as an important virulence factor. However, there is little information on the structure and collagenolytic ...mechanism of Vibrio collagenase. Here, we report the crystal structure of the collagenase module (CM) of Vibrio collagenase VhaC and the conformation of VhaC in solution. Structural and biochemical analyses and molecular dynamics studies reveal that triple-helical collagen is initially recognized by the activator domain, followed by subsequent cleavage by the peptidase domain along with the closing movement of CM. This is different from the peptidolytic mode or the proposed collagenolysis of Clostridium collagenase. We propose a model for the integrated collagenolytic mechanism of VhaC, integrating the functions of VhaC accessory domains and its collagen degradation pattern. This study provides insight into the mechanism of bacterial collagenolysis and helps in structure-based drug design targeting of the Vibrio collagenase.
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•Fatty acid-derived hydrocarbons are ideal alternatives to petroleum-based fuels.•A growing number of hydrocarbon biosynthetic systems have been discovered.•Diverse fatty acid-derived ...hydrocarbon synthases share common precursors.•Elucidated mechanisms indicate diverse hydrocarbon synthases follow similar chemical logic.•Great efforts have been made on enzyme engineering of hydrocarbon synthases.
Enormous fuel consumption and growing environmental concerns have been spurring development of renewable biofuels. Among various biofuels, fatty acid-derived biohydrocarbons are ideal alternatives to non-renewable fossil fuels due to their closest properties to petroleum-based fuels. In the past decade, novel hydrocarbon-producing enzymes have continuously been discovered and engineered. Here, we review the recent advances in biosynthesis of fatty acid-derived hydrocarbons with emphasis on enzymology and enzyme engineering, based on which some outlooks are provided.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Cytochrome P450 enzymes are capable of catalyzing a great variety of synthetically useful reactions such as selective C–H functionalization. Surrogate redox partners are widely used for ...reconstitution of P450 activity based on the assumption that the choice of these auxiliary proteins or their mode of action does not affect the type and selectivity of reactions catalyzed by P450s. Herein, we present an exceptional example to challenge this postulate. MycG, a multifunctional biosynthetic P450 monooxygenase responsible for hydroxylation and epoxidation of 16-membered ring macrolide mycinamicins, is shown to catalyze the unnatural N-demethylation(s) of a range of mycinamicin substrates when partnered with the free Rhodococcus reductase domain RhFRED or the engineered Rhodococcus-spinach hybrid reductase RhFRED-Fdx. By contrast, MycG fused with the RhFRED or RhFRED-Fdx reductase domain mediates only physiological oxidations. This finding highlights the larger potential role of variant redox partner protein–protein interactions in modulating the catalytic activity of P450 enzymes.
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IJS, KILJ, NUK, PNG, UL, UM
An efficient strategy was developed for the rapid separation and enrichment of bafilomycin A1 (baf A1) from a crude extract of the marine microorganism
fermentation. This strategy comprises ...liquid-liquid extraction (LLE) with a three-phase solvent system (
-hexane-ethyl acetate-acetonitrile-water = 7:3:5:5,
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) followed by separation using high-speed counter-current chromatography (HSCCC). The results showed that a 480.2-mg fraction of baf A1-enriched extract in the middle phase of the three-phase solvent system was prepared from 4.9 g of crude extract after two consecutive one-step operations. Over 99% of soybean oil, the main hydrophobic waste in the crude extract, and the majority of hydrophilic impurities were distributed in the upper and lower phase, respectively. HSCCC was used with a two-phase solvent system composed of
-hexane-acetonitrile-water (15:8:12,
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) to isolate and purify baf A1 from the middle phase fraction, which yielded 77.4 mg of baf A1 with > 95% purity within 90 min. The overall recovery of baf A1 in the process was determined to be 95.7%. The use of a three-phase solvent system represents a novel strategy for the simultaneous removal of hydrophobic oil and hydrophilic impurities from a microbial fermentation extract.
This study investigates the enhanced effect of timber infill walls on the seismic behavior of traditional Chinese timber frames. Two 1/2 scaled traditional Chinese timber infill walls (TIWs), two 1/2 ...scaled timber frames with timber infill walls (TFTIWs) and one 1/2 scaled timber frame (TF) were fabricated and tested under low-cyclic reversed loading. The failure modes, strength, stiffness, and energy consumption capacity of the TIWs and the TFTIWs were obtained, and the effects of the TIWs on the seismic performance of the TFTIWs were investigated. The results showed that the TIWs can increase the stiffness and ultimate bearing capacity of the TF, up to 60% and 80%, respectively. The strength degradation coefficient of the TFTIWs was slightly higher than that of the TF when the inter-story drift ratio exceeded 0.02 rad, and the TIWs helped to mitigate the strength degradation of the TFTIWs. It was also found that the TFTIWs had a higher cumulative energy dissipation when compared with the TF (up to a 60% increase), indicating the TIWs had reasonably good energy dissipation capacity. When the TIWs generated a solid contribution to the carrying capacity and energy dissipation of the TF, the lateral drift thresholds were 1/100 and 1/43 of the column height, respectively. Furthermore, the TIWs and TFTIWs presented a good ductility, and the TIW could effectively reduce the pull-out amount of the tenon from the mortise of the TF; however, the TIWs had little influence on the stiffness degradation level or improvement of the ductility of the TF.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Traditional building energy evaluation methods usually adopt a single energy use indicator as the benchmark. However, such single-indicator based methods often lead to a paradox that switching off ...building service equipment or systems is equivalent to energy saving. To avoid such paradox for a more pragmatic and comprehensive evaluation of building energy use, this paper aims to establish a robust evaluation model of the operational energy efficiency for public buildings by coupling the analytic hierarchy process (AHP) and fuzzy comprehensive evaluation (FCE). Firstly, 23 quantitative indexes and 7 qualitative indexes are extracted from existing building energy codes/standards in the input-output perspective of energy performance evaluations for public buildings. The AHP is then used to determine the weight of each index based on a proposed index correction method, which extends the range of applicability of the index system. Finally, a three-level comprehensive evaluation model is established for the operational energy efficiency of public buildings. Furthermore, a case study is conducted on a typical office building to validate the evaluation results with the proposed model. The case building achieves an overall score of 84.9 at a “Good” level, and corresponding recommendations are made for further improving building performance indexes. The proposed method is of great significance to developing building energy efficiency assessment systems and guidelines in China and over the world.
•A comprehensive evaluation system of building energy efficiency is established.•The evaluation method of building energy efficiency is based on energy input-output.•Analytic hierarchy process combined with fuzzy comprehensive evaluation method is adopted.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Notoamides produced by Aspergillus spp. bearing the bicyclo2.2.2diazaoctane core structure with unusual structural diversity represent a compelling system to understand the biosynthesis of fungal ...prenylated indole alkaloids. Herein, we report the in vitro characterization of NotB, which catalyzes the indole 2,3-oxidation of notoamide E (13), leading to notoamides C (11) and D (12) through an apparent pinacol-like rearrangement. This unique enzymatic reaction with high substrate specificity, together with the information derived from precursor incorporation experiments using 13C2–15N2 quadruply labeled notoamide S (10), demonstrates 10 as a pivotal branching point in notoamide biosynthesis.
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IJS, KILJ, NUK, PNG, UL, UM
α-Galactosidases are broadly used in feed, food, chemical, pulp, and pharmaceutical industries. However, there lacks a satisfactory microbial cell factory that is able to produce α-galactosidases ...efficiently and cost-effectively to date, which prevents these important enzymes from greater application. In this study, the secretory expression of an Aspergillus niger α-galactosidase (AGA) in Pichia pastoris was systematically investigated. Through codon optimization, signal peptide replacement, comparative selection of host strain, and saturation mutagenesis of the P1' residue of Kex2 protease cleavage site for efficient signal peptide removal, a mutant P. pastoris KM71H (Muts) strain of AGA-I with the specific P1' site substitution (Glu to Ile) demonstrated remarkable extracellular α-galactosidase activity of 1299 U/ml upon a 72 h methanol induction in 2.0 L fermenter. The engineered yeast strain AGA-I demonstrated approximately 12-fold higher extracellular activity compared to the initial P. pastoris strain. To the best of our knowledge, this represents the highest yield and productivity of a secreted α-galactosidase in P. pastoris, thus holding great potential for industrial application.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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