Deciphering the stereo-specific catalytic mechanisms of cis-epoxysuccinate hydrolases producing L(+)-tartaric acid.

Microbial epoxide hydrolases, cis-epoxysuccinate hydrolases (CESHs), have been utilized for commercial production of enantiomerically pure L(+)- and D(-)-tartaric acids for decades. However, the stereo-catalytic mechanism of CESH producing L(+)-tartaric acid (CESH[L]) remains unclear. Herein, the crystal structures of two CESH[L]s in ligand-free, product-complexed, and catalytic intermediate forms were determined. These structures revealed the unique specific binding mode for the mirror-symmetric substrate, an active catalytic triad consisting of Asp-His-Glu, and an arginine providing a proton to the oxirane oxygen to facilitate the epoxide ring-opening reaction, which has been pursued for decades. These results provide the structural basis for the rational engineering of these industrial biocatalysts.

Composition of Lignocellulose Hydrolysate in Different Biorefinery Strategies: Nutrients and Inhibitors.

The hydrolysis and biotransformation of lignocellulose, i.e., biorefinery, can provide human beings with biofuels, bio-based chemicals, and materials, and is an important technology to solve the fossil energy crisis and promote global sustainable development. Biorefinery involves steps such as pretreatment, saccharification, and fermentation, and researchers have developed a variety of biorefinery strategies to optimize the process and reduce process costs in recent years. Lignocellulosic hydrolysates are platforms that connect the saccharification process and downstream fermentation. The hydrolysate composition is closely related to biomass raw materials, the pretreatment process, and the choice of biorefining strategies, and provides not only nutrients but also possible inhibitors for downstream fermentation. In this review, we summarized the effects of each stage of lignocellulosic biorefinery on nutrients and possible inhibitors, analyzed the huge differences in nutrient retention and inhibitor generation among various biorefinery strategies, and emphasized that all steps in lignocellulose biorefinery need to be considered comprehensively to achieve maximum nutrient retention and optimal control of inhibitors at low cost, to provide a reference for the development of biomass energy and chemicals.

Integrated lactic acid production from lignocellulosic agricultural wastes under thermal conditions.

The production of lactic acid (LA) from agricultural wastes attracts great attention because of the sustainability and abundance of lignocellulosic feedstocks, as well as the increasing demand for biodegradable polylactic acid. In this study, we isolated a thermophilic strain Geobacillus stearothermophilus 2H-3 for use in robust production of L-(+)LA under the optimal conditions of 60 °C, pH 6.5, which were consistent with the whole-cell-based consolidated bio-saccharification (CBS) process. Sugar-rich CBS hydrolysates derived from various agricultural wastes, including corn stover, corncob residue, and wheat straw, were used as the carbon sources for 2H-3 fermentation by directly inoculating 2H-3 cells into the CBS system, without intermediate sterilization, nutrient supplementation, or adjustment of fermentation conditions. Thus, we successfully combined two whole-cell-based steps into a one-pot successive fermentation process to efficiently produce LA with high optical purity (99.5%), titer (51.36 g/L), and yield (0.74 g/gbiomass). This study provides a promising strategy for LA production from lignocellulose through CBS and 2H-3 fermentation integration.

Hydrogenase and Nitrogenase: Key Catalysts in Biohydrogen Production.

Hydrogen with high energy content is considered to be a promising alternative clean energy source. Biohydrogen production through microbes provides a renewable and immense hydrogen supply by utilizing raw materials such as inexhaustible natural sunlight, water, and even organic waste, which is supposed to solve the two problems of "energy supply and environment protection" at the same time. Hydrogenases and nitrogenases are two classes of key enzymes involved in biohydrogen production and can be applied under different biological conditions. Both the research on enzymatic catalytic mechanisms and the innovations of enzymatic techniques are important and necessary for the application of biohydrogen production. In this review, we introduce the enzymatic structures related to biohydrogen production, summarize recent enzymatic and genetic engineering works to enhance hydrogen production, and describe the chemical efforts of novel synthetic artificial enzymes inspired by the two biocatalysts. Continual studies on the two types of enzymes in the future will further improve the efficiency of biohydrogen production and contribute to the economic feasibility of biohydrogen as an energy source.

Artificial Small Molecules as Cofactors and Biomacromolecular Building Blocks in Synthetic Biology: Design, Synthesis, Applications, and Challenges.

Enzymes are essential catalysts for various chemical reactions in biological systems and often rely on metal ions or cofactors to stabilize their structure or perform functions. Improving enzyme performance has always been an important direction of protein engineering. In recent years, various artificial small molecules have been successfully used in enzyme engineering. The types of enzymatic reactions and metabolic pathways in cells can be expanded by the incorporation of these artificial small molecules either as cofactors or as building blocks of proteins and nucleic acids, which greatly promotes the development and application of biotechnology. In this review, we summarized research on artificial small molecules including biological metal cluster mimics, coenzyme analogs (mNADs), designer cofactors, non-natural nucleotides (XNAs), and non-natural amino acids (nnAAs), focusing on their design, synthesis, and applications as well as the current challenges in synthetic biology.

Regiodivergent and Enantioselective Hydroxylation of C-H bonds by Synergistic Use of Protein Engineering and Exogenous Dual-Functional Small Molecules.

It is a great challenge to optionally access diverse hydroxylation products from a given substrate bearing multiple reaction sites of sp3 and sp2 C-H bonds. Herein, we report the highly selective divergent hydroxylation of alkylbenzenes by an engineered P450 peroxygenase driven by a dual-functional small molecule (DFSM). Using combinations of various P450BM3 variants with DFSMs enabled access to more than half of all possible hydroxylated products from each substrate with excellent regioselectivity (up to >99 %), enantioselectivity (up to >99 % ee), and high total turnover numbers (up to 80963). Crystal structure analysis, molecular dynamic simulations, and theoretical calculations revealed that synergistic effects between exogenous DFSMs and the protein environment controlled regio- and enantioselectivity. This work has implications for exogenous-molecule-modulated enzymatic regiodivergent and enantioselective hydroxylation with potential applications in synthetic chemistry.

Engineering Cytochrome P450BM3 Enzymes for Direct Nitration of Unsaturated Hydrocarbons.

Applications of the peroxidase activity of cytochrome P450 enzymes in synthetic chemistry remain largely unexplored. We present herein a protein engineering strategy to increase cytochrome P450BM3 peroxidase activity for the direct nitration of aromatic compounds and terminal aryl-substituted olefins in the presence of a dual-functional small molecule (DFSM). Site-directed mutations of key active-site residues allowed the efficient regulation of steric effects to limit substrate access and, thus, a significant decrease in monooxygenation activity and increase in peroxidase activity. Nitration of several phenol and aniline compounds also yielded ortho- and para-nitration products with moderate-to-high total turnover numbers. Besides direct aromatic nitration by P450 variants using nitrite as a nitrating agent, we also demonstrated the use of the DFSM-facilitated P450 peroxidase system for the nitration of the vinyl group of styrene and its derivatives.

Structural basis for the DNA-binding activity of human ARID4B Tudor domain.

Human ARID4A and ARID4B are homologous proteins that are important in controlling gene expression and epigenetic regulation but have distinct functions. Previous studies have shown that the N-terminal domain of ARID4A is an unusual interdigitated double Tudor domain with DNA-binding activity. However, how the Tudor domain of ARID4B differs from that of ARID4A remains unknown. Here, we found that the ARID4B Tudor domain has significantly weaker DNA affinity than the ARID4A Tudor domain despite sharing more than 80% sequence identity. Structure determination and DNA titration analysis indicated that the ARID4B Tudor domain is also an interdigitated double Tudor domain with a DNA-binding surface similar to ARID4A. We identified a residue close to the DNA-binding site of the Tudor domain that differs between ARID4A and ARID4B. The Leu50 in ARID4A is Glu50 in ARID4B, and the latter forms salt bridges with two lysine residues at the DNA-binding surface. This causes a decrease in the strength of positive charge, thus reducing DNA-binding affinity while significantly increasing protein stability. We also found that a C-terminal extension region enhances the DNA-binding affinity of the ARID4B Tudor domain. This C-terminal extension is disordered and contains a positively charged RGR motif, providing an additional DNA-binding site. Finally, sequence and phylogenetic analyses indicated that the residue differences and the presence of the RGR extension region are conserved. These results provide new insight into the functional differences between ARID4A and ARID4B proteins, as well as elucidating the function of the disordered regions in these proteins.

Diversification of phenolic glucosides by two UDP-glucosyltransferases featuring complementary regioselectivity.

BACKGROUND: Glucoside natural products have been showing great medicinal values and potentials. However, the production of glucosides by plant extraction, chemical synthesis, and traditional biotransformation is insufficient to meet the fast-growing pharmaceutical demands. Microbial synthetic biology offers promising strategies for synthesis and diversification of plant glycosides. RESULTS: In this study, the two efficient UDP-glucosyltransferases (UGTs) (UGT85A1 and RrUGT3) of plant origin, that are capable of recognizing phenolic aglycons, are characterized in vitro. The two UGTs show complementary regioselectivity towards the alcoholic and phenolic hydroxyl groups on phenolic substrates. By combining a developed alkylphenol bio-oxidation system and these UGTs, twenty-four phenolic glucosides are enzymatically synthesized from readily accessible alkylphenol substrates. Based on the bio-oxidation and glycosylation systems, a number of microbial cell factories are constructed and applied to biotransformation, giving rise to a variety of plant and plant-like O-glucosides. Remarkably, several unnatural O-glucosides prepared by the two UGTs demonstrate better prolyl endopeptidase inhibitory and/or anti-inflammatory activities than those of the clinically used glucosidic drugs including gastrodin, salidroside and helicid. Furthermore, the two UGTs are also able to catalyze the formation of N- and S-glucosidic bonds to produce N- and S-glucosides. CONCLUSIONS: Two highly efficient UGTs, UGT85A1 and RrUGT3, with distinct regioselectivity were characterized in this study. A group of plant and plant-like glucosides were efficiently synthesized by cell-based biotransformation using a developed alkylphenol bio-oxidation system and these two UGTs. Many of the O-glucosides exhibited better PEP inhibitory or anti-inflammatory activities than plant-origin glucoside drugs, showing significant potentials for new glucosidic drug development.

Structural basis of X chromosome DNA recognition by the MSL2 CXC domain during Drosophila dosage compensation.

The male-specific lethal dosage compensation complex (MSL-DCC) selectively assembles on the X chromosome in Drosophila males and activates gene transcription by twofold through histone acetylation. An MSL recognition element (MRE) sequence motif nucleates the initial MSL association, but how it is recognized remains unknown. Here, we identified the CXC domain of MSL2 specifically recognizing the MRE motif and determined its crystal structure bound to specific and nonspecific DNAs. The CXC domain primarily contacts one strand of DNA duplex and employs a single arginine to directly read out dinucleotide sequences from the minor groove. The arginine is flexible when bound to nonspecific sequences. The core region of the MRE motif harbors two binding sites on opposite strands that can cooperatively recruit a CXC dimer. Specific DNA-binding mutants of MSL2 are impaired in MRE binding and X chromosome localization in vivo. Our results reveal multiple dynamic DNA-binding modes of the CXC domain that target the MSL-DCC to X chromosomes.

The chaperone Hsp70 is a BH3 receptor activated by the pro-apoptotic Bim to stabilize anti-apoptotic clients.

The chaperone heat shock protein 70 (Hsp70) is crucial for avoiding protein misfolding under stress, but is also up-regulated in many kinds of cancers, where its ability to buffer cellular stress prevents apoptosis. Previous research has suggested Hsp70 interacts with pro-apoptotic Bcl-2 family proteins, including Bim and Bax. However, a definitive demonstration of this interaction awaits, and insights into the structural basis and molecular mechanism remain unclear. Earlier studies have identified a Bcl-2 homology 3 (BH3) domain present in Bcl-2 family members that engages receptors to stimulate apoptosis. We now show that Hsp70 physically interacts with pro-apoptotic multidomain and BH3-only proteins via a BH3 domain, thereby serving as a novel BH3 receptor, using in vitro fluorescent polarization (FP), isothermal titration calorimetry (ITC), and cell-based co-immunoprecipitation (co-IP) experiments, 1H-15N-transverse relaxation optimized spectroscopy (TROSY-HSQC), trypsin proteolysis, ATPase activity, and denatured rhodanese aggregation measurements further demonstrated that BimBH3 binds to a novel allosteric site in the nucleotide-binding domain (NBD) of Hsp70, by which Bim acts as a positive co-chaperone to promote the ATPase activity and chaperone functions. A dual role of Hsp70's anti-apoptotic function was revealed that when it keeps Bim in check to inhibit apoptosis, it simultaneously stabilizes oncogenic clients including AKT and Raf-1 with the aid of Bim. Two faces of Bim in cell fate regulation were revealed that in opposite to its well-established pro-apoptotic activator role, Bim could help the folding of oncogenic proteins.

Structural Basis for Selective Oxidation of Phosphorylated Ethylphenols by Cytochrome P450 Monooxygenase CreJ.

Selective oxidation of C-H bonds in alkylphenols holds great significance for not only structural derivatization in pharma- and biomanufacturing but also biological degradation of these toxic chemicals in environmental protection. A unique chemomimetic biocatalytic system using enzymes from a p-cresol biodegradation pathway has recently been developed. As the central biocatalyst, the cytochrome P450 monooxygenase CreJ oxidizes diverse p- and m-alkylphenol phosphates with perfect stereoselectivity at different efficiencies. However, the mechanism of regio- and stereoselectivity of this chemomimetic biocatalytic system remained unclear. Here, using p- and m-ethylphenol substrates, we elucidate the CreJ-catalyzed key steps for selective oxidations. The crystal structure of CreJ in complex with m-ethylphenol phosphate was solved and compared with its complex structure with p-ethylphenol phosphate isomer. The results indicate that the conformational changes of substrate-binding residues are slight, while the substrate promiscuity is achieved mainly by the available space in the catalytic cavity. Moreover, the catalytic preferences of regio- and stereoselective hydroxylation for the two ethylphenol substrates is explored by molecular dynamics simulations. The ethyl groups in the complexes display different flexibilities, and the distances of the active oxygen to H pro-S and H pro-R of methylene agree with the experimental stereoselectivity. The regioselectivity can be explained by the distances and bond dissociation energy. These results provide not only the mechanistic insights into CreJ regio- and stereoselectivity but also the structural basis for further P450 enzyme design and engineering.IMPORTANCE The key cytochrome P450 monooxygenase CreJ showed excellent regio- and stereoselectivity in the oxidation of various alkylphenol substrates. C-H bond functionalization of these toxic alkylphenols holds great significance for both biological degradation of these environmental chemicals and production of value-added structural derivatives in pharmaceutical and biochemical industries. Our results, combined with in vitro enzymatic assays, crystal structure determination of enzyme-substrate complex, and molecular dynamics simulations, provide not only significant mechanism elucidation of the regio- and stereoselective catalyzation mediated by CreJ but also the promising directions for future engineering efforts of this enzyme toward more useful products. It also has great extendable potential to couple this multifunctional P450 enzyme with other biocatalysts (e.g., hydroxyl-based glycosylase) to access more alkylphenol-derived high-value chemicals through environment-friendly biocatalysis and biotransformation.

Alternative σI/anti-σI factors represent a unique form of bacterial σ/anti-σ complex.

The σ70 family alternative σI factors and their cognate anti-σI factors are widespread in Clostridia and Bacilli and play a role in heat stress response, virulence, and polysaccharide sensing. Multiple σI/anti-σI factors exist in some lignocellulolytic clostridial species, specifically for regulation of components of a multienzyme complex, termed the cellulosome. The σI and anti-σI factors are unique, because the C-terminal domain of σI (SigIC) and the N-terminal inhibitory domain of anti-σI (RsgIN) lack homology to known proteins. Here, we report structure and interaction studies of a pair of σI and anti-σI factors, SigI1 and RsgI1, from the cellulosome-producing bacterium, Clostridium thermocellum. In contrast to other known anti-σ factors that have N-terminal helical structures, RsgIN has a ß-barrel structure. Unlike other anti-σ factors that bind both σ2 and σ4 domains of the σ factors, RsgIN binds SigIC specifically. Structural analysis showed that SigIC contains a positively charged surface region that recognizes the promoter -35 region, and the synergistic interactions among multiple interfacial residues result in the specificity displayed by different σI/anti-σI pairs. We suggest that the σI/anti-σI factors represent a distinctive mode of σ/anti-σ complex formation, which provides the structural basis for understanding the molecular mechanism of the intricate σI/anti-σI system.

Structural Basis of Specificity for Carboxyl-Terminated Acyl Donors in a Bacterial Acyltransferase.

Macrolactins (MLNs) are a class of important antimacular degeneration and antitumor agents. Malonylated/succinylated MLNs are even more important due to their efficacy in overcoming multi-drug-resistant bacteria. However, which enzyme catalyzes this reaction remains enigmatic. Herein, we deciphered a ß-lactamase homologue BmmI to be responsible for this step. BmmI could specifically attach C3-C5 alkyl acid thioesters onto 7-OH of MLN A and also exhibits substrate promiscuity toward acyl acceptors with different scaffolds. The crystal structure of BmmI covalently linked to the succinyl group and systematic mutagenesis highlighted the role of oxyanion holelike geometry in the recognition of carboxyl-terminated acyl donors. The engineering of this geometry expanded its substrate scope, with the R166A/G/Q variants recognizing up to C12 alkyl acid thioester. The structure of BmmI with acyl acceptor MLN A revealed the importance of Arg292 in the recognition of macrolide substrates. Moreover, the mechanism of the BmmI-catalyzed acyltransfer reaction was established, unmasking the deft role of Lys76 in governing acyl donors as well as catalysis. Our studies uncover the delicate mechanism underlying the substrate selectivity of acyltransferases, which would guide rational enzyme engineering for drug development.

Selective oxidation of aliphatic C-H bonds in alkylphenols by a chemomimetic biocatalytic system.

Selective oxidation of aliphatic C-H bonds in alkylphenols serves significant roles not only in generation of functionalized intermediates that can be used to synthesize diverse downstream chemical products, but also in biological degradation of these environmentally hazardous compounds. Chemo-, regio-, and stereoselectivity; controllability; and environmental impact represent the major challenges for chemical oxidation of alkylphenols. Here, we report the development of a unique chemomimetic biocatalytic system originated from the Gram-positive bacterium Corynebacterium glutamicum The system consisting of CreHI (for installation of a phosphate directing/anchoring group), CreJEF/CreG/CreC (for oxidation of alkylphenols), and CreD (for directing/anchoring group offloading) is able to selectively oxidize the aliphatic C-H bonds of p- and m-alkylated phenols in a controllable manner. Moreover, the crystal structures of the central P450 biocatalyst CreJ in complex with two representative substrates provide significant structural insights into its substrate flexibility and reaction selectivity.

Solution structure of a unicellular microalgae-derived translationally controlled tumor protein revealed both conserved features and structural diversity.

Translationally controlled tumor proteins (TCTPs) are eukaryote-conserved multifunctional proteins, but their primary functions are not well understood yet. Study on TCTP from unicellular species may provide insight into the primary function of the TCTP family. Bioinformatic analysis indicated that the TCTP from Nannochloropsis oceanica (NoTCTP), a model unicellular microalga for biodiesel and polyunsaturated fatty acid production, has low sequence homology to other structure-known TCTPs and two TCTP signature patterns are not detected in NoTCTP. Hence, we determined the solution structure of NoTCTP. The overall structure of NoTCTP, including a long flexible loop, a ß-barrel subdomain, and a helical subdomain, is generally similar to other TCTP structures. NoTCTP has a eukaryote-conserved surface for the binding of eukaryotic elongation factor 1B, confirming that TCTP is involved in protein synthesis, which is one of the primary functions of TCTP. Additionally, NoTCTP has distinct features different from other TCTPs. NoTCTP structure lacks a short α-helix which exists in all other known TCTP structures. The helical subdomain and some loops of NoTCTP also have distinct conformations among the TCTP family proteins. Therefore, our study on NoTCTP revealed not only conserved structural features but also the structural diversity of TCTP family proteins.

An Effective Strategy for Identification of Highly Unstable Bacillaenes.

Exploration of unstable compounds is a rarely explored area of natural product research. We describe the integration of genomic and metabolomic analyses with bioassay-guided compound mining to effectively explore unstable bacillaenes. New bacillaene structures (2, 4, and 5) were identified from compound mixtures using the DANS-SVI (differential analysis of 2D NMR spectrum-single spectrum with variable intensities) method, which were further verified by the isolation of the pure compounds under strictly controlled conditions. Compound 1 exhibited antibacterial activity against multi-drug-resistant bacterial strains, while glycosylation decreased the activity of the bacillaene scaffold.

Enantiomeric Tartaric Acid Production Using cis-Epoxysuccinate Hydrolase: History and Perspectives.

Tartaric acid is an important chiral chemical building block with broad industrial and scientific applications. The enantioselective synthesis of l(+)- and d(-)-tartaric acids has been successfully achieved using bacteria presenting cis-epoxysuccinate hydrolase (CESH) activity, while the catalytic mechanisms of CESHs were not elucidated clearly until very recently. As biocatalysts, CESHs are unique epoxide hydrolases because their substrate is a small, mirror-symmetric, highly hydrophilic molecule, and their products show very high enantiomeric purity with nearly 100% enantiomeric excess. In this paper, we review over forty years of the history, process and mechanism studies of CESHs as well as our perspective on the future research and applications of CESH in enantiomeric tartaric acid production.

Solution structure of an archaeal DUF61 family protein SSO0941 encoded by a gene in the operon of box C/D RNA protein complexes.

Domain of unknown function 61 (DUF61) family proteins widely exist in archaea and the genes of DUF61 proteins in crenarchaea are in an operon containing two genes of box C/D RNA protein complexes. Here we report the solution NMR structure of DUF61 family member protein SSO0941, from the hyperthermophilic archaeon Sulfolobus solfataricus. SSO0941 has a rigid core structure and flexible N- and C-terminal regions as well as a negatively-charged independent C-terminal helix. The core structure consists of N- and C-terminal subdomains, in which the C-terminal subdomain shows significant structural similarity with several nucleic acid binding proteins. The structure of SSO0941 is the first representative structure of DUF61 family proteins.