Unveiling the repressive mechanism of a PPS-like regulator (PspR) in polyhydroxyalkanoates biosynthesis network.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is a type of polyhydroxyalkanoates (PHA) that exhibits numerous outstanding properties and is naturally synthesized and elaborately regulated in various microorganisms. However, the regulatory mechanism involving the specific regulator PhaR in Haloferax mediterranei, a major PHBV production model among Haloarchaea, is not well understood. In our previous study, we showed that deletion of the phosphoenolpyruvate (PEP) synthetase-like (pps-like) gene activates the cryptic phaC genes in H. mediterranei, resulting in enhanced PHBV accumulation. In this study, we demonstrated the specific function of the PPS-like protein as a negative regulator of phaR gene expression and PHBV synthesis. Chromatin immunoprecipitation (ChIP), in situ fluorescence reporting system, and in vitro electrophoretic mobility shift assay (EMSA) showed that the PPS-like protein can bind to the promoter region of phaRP. Computational modeling revealed a high structural similarity between the rifampin phosphotransferase (RPH) protein and the PPS-like protein, which has a conserved ATP-binding domain, a His domain, and a predicted DNA-binding domain. Key residues within this unique DNA-binding domain were subsequently validated through point mutation and functional evaluations. Based on these findings, we concluded that PPS-like protein, which we now renamed as PspR, has evolved into a repressor capable of regulating the key regulator PhaR, and thereby modulating PHBV synthesis. This regulatory network (PspR-PhaR) for PHA biosynthesis is likely widespread among haloarchaea, providing a novel approach to manipulate haloarchaea as a production platform for high-yielding PHA. KEY POINTS: • The repressive mechanism of a novel inhibitor PspR in the PHBV biosynthesis was demonstrated • PspR is widespread among the PHA accumulating haloarchaea • It is the first report of functional conversion from an enzyme to a trans-acting regulator in haloarchaea.

Computational redesign of enzymes for regio- and enantioselective hydroamination.

Introduction of innovative biocatalytic processes offers great promise for applications in green chemistry. However, owing to limited catalytic performance, the enzymes harvested from nature's biodiversity often need to be improved for their desired functions by time-consuming iterative rounds of laboratory evolution. Here we describe the use of structure-based computational enzyme design to convert Bacillus sp. YM55-1 aspartase, an enzyme with a very narrow substrate scope, to a set of complementary hydroamination biocatalysts. The redesigned enzymes catalyze asymmetric addition of ammonia to substituted acrylates, affording enantiopure aliphatic, polar and aromatic ß-amino acids that are valuable building blocks for the synthesis of pharmaceuticals and bioactive compounds. Without a requirement for further optimization by laboratory evolution, the redesigned enzymes exhibit substrate tolerance up to a concentration of 300 g/L, conversion up to 99%, ß-regioselectivity >99% and product enantiomeric excess >99%. The results highlight the use of computational design to rapidly adapt an enzyme to industrially viable reactions.

Biochemical and structural characterization of a highly active branched-chain amino acid aminotransferase from Pseudomonas sp. for efficient biosynthesis of chiral amino acids.

Aminotransferases (ATs) are important biocatalysts for the synthesis of chiral amines because of their capability of introducing amino group into ketones or keto acids as well as their high enantioselectivity, high regioselectivity. Among all ATs, branched-chain amino acid aminotransferase (BCAT) can use branched-chain amino acids (BCAAs) as substrate, including L-valine, L-leucine, and L-isoleucine, with α-ketoglutarate to form the corresponding α-keto acids and L-glutamate. Alternatively, BCATs have been used for the biosynthesis of unnatural amino acids, such as L-tert-leucine and L-norvaline. In the present study, the BCAT from Pseudomonas sp. (PsBCAT) was cloned and expressed in Escherichia coli for biochemical and structural analyses. The optimal reaction temperature and pH of PsBCAT were 40 °C and 8.5, respectively. PsBCAT exhibited a comparatively broader substrate spectrum and showed remarkably high activity with bulked aliphatic L-amino acids (kcat up to 220 s-1). Additionally, PsBCAT had activities with aromatic L-amino acids, L-histidine, L-lysine, and L-threonine. This substrate promiscuity is unique for the BCAT family and could prove useful in industrial applications. To analyze the catalytic mechanism of PsBCAT with the broad substrate spectrum, the crystal structure of PsBCAT was also determined. Based on the determined crystal structure, we found some differences in the organization of the substrate binding cavity, which may influence the substrate specificity of the enzyme. Finally, conjugated with the ornithine aminotransferase (OrnAT) to shift the reaction equilibrium towards the product formation, the coupled system was applied to the asymmetric synthesis of L-tert-leucine and L-norvaline. In summary, the structural and functional characteristics of PsBCAT were analyzed in detail, and this information will be conducive to industrial production of enantiopure chiral amino acids by aminotransferase.

Engineering improved thermostability of the GH11 xylanase from Neocallimastix patriciarum via computational library design.

Xylanases, which cleave the ß-1,4-glycosidic bond between xylose residues to release xylooligosaccharides (XOS), are widely used as food additives, animal feeds, and pulp bleaching agents. However, the thermally unstable nature of xylanases would hamper their industrial application. In this study, we used in silico design in a glycoside hydrolase family (GH) 11 xylanase to stabilize the enzyme. A combination of the best mutations increased the apparent melting temperature by 14 °C and significantly enhanced thermostability and thermoactivation. The variant also showed an upward-shifted optimal temperature for catalysis without compromising its activity at low temperatures. Moreover, a 10-fold higher XOS production yield was obtained at 70 °C, which compensated the low yield obtained with the wild-type enzyme. Collectively, the variant constructed by the computational strategy can be used as an efficient biocatalyst for XOS production at industrially viable conditions.

Structural features and dynamic investigations of the membrane-bound cytochrome P450 17A1.

Cytochrome P450 (CYP) 17A1 is a dual-function monooxygenase with a critical role in the synthesis of many human steroid hormones. The enzyme is an important target for treatment of breast and prostate cancers that proliferate in response to estrogens and androgens. Despite the crystallographic structures available for CYP17A1, no membrane-bound structural features of this enzyme at atomic level are available. Accumulating evidence has indicated that the interactions between bounded CYPs and membrane could contribute to the recruitment of lipophilic substrates. To this end, we have investigated the effects on structural characteristics in the presence of the membrane for CYP17A1. The MD simulation results demonstrate a spontaneous insertion process of the enzyme to the lipid. Two predominant modes of CYP17A1 in the membrane are captured, characterized by the depths of insertion and orientations of the enzyme to the membrane surface. The measured heme tilt angles show good consistence with experimental data, thereby verifying the validity of the structural models. Moreover, conformational changes induced by the membrane might have impact on the accessibility of the active site to lipophilic substrates. The dynamics of internal aromatic gate formed by Trp220 and Phe224 are suggested to regulate tunnel opening motions. The knowledge of the membrane binding characteristics could guide future experimental and computational works on membrane-bound CYPs so that various investigations of CYPs in their natural, lipid environment rather than in artificially solubilized forms may be achieved.

Molecular dynamics investigations of regioselectivity of anionic/aromatic substrates by a family of enzymes: a case study of diclofenac binding in CYP2C isoforms.

The CYP2C subfamily is of particular importance in the metabolism of drugs, food toxins, and procarcinogens. Like other P450 subfamilies, 2C enzymes share a high sequence identity, but significantly contribute in different ways to hepatic capacity to metabolize drugs. They often metabolize the same substrate to more than one product with different catalytic sites. Because it is challenging to characterize experimentally, much still remains unknown about the reason for why the substrate regioselectivity of these closely related subfamily members is different. Here, we have investigated the structural features of CYP2C8, CYP2C9, and CYP2C19 bound with their shared substrate diclofenac to elucidate the underlying molecular mechanism for the substrate regioselectivity of CYP2C subfamily enzymes. The obtained results demonstrate how a sequence divergence for the active site residues causes heterogeneous variations in the secondary structures and in major tunnel selections, and further affects the shape and chemical properties of the substrate-binding site. Structural analysis and free energy calculations showed that the most important determinants of regioselectivity among the CYP2C isoforms are the geometrical features of the active sites, as well as the hydrogen bonds and the hydrophobic interactions, mainly presenting as the various locations of Arg108 and substitutions of Phe205 for Ile205 in CYP2C8. The MM-GB/SA calculations combined with PMF results accord well with the experimental KM values, bridging the gap between the theory and the experimentally observed results of binding affinity differences. The present study provides important insights into the structure-function relationships of CYP2C subfamily enzymes, the knowledge of ligand binding characteristics and key residue contributions could guide future experimental and computational work on the synthesis of drugs with better pharmacokinetic properties so that CYP interactions could be avoided.

Molecular basis of the recognition of arachidonic acid by cytochrome P450 2E1 along major access tunnel.

Cytochrome P450 2E1 is widely known for its ability to oxidize both low molecular weight xenobiotics and endogenous fatty acids (e.g., arachidonic acid (AA)). In this study, we investigated the structural features of the AA-bound CYP2E1 complex utilizing molecular dynamics (MD) and found that the distinct binding modes for both AA and fatty acid analog are conserved. Moreover, multiple random acceleration MD simulations and steered MD simulations uncovered the most possible tunnel for fatty acids. The main attractions are derived from three key residues, His107, Ala108, and His109, whose side chains reorient to keep ligands bound via hydrogen bonds during the initial unbinding process. More importantly, based on the calculated binding free energy results, we hypothesize that the hydrogen bonds between the receptor and the ligand are the most important contributors involved in the binding affinity. Thus, it is inferred that the hydrogen bonds between these three residues and the ligand may help offer insights into the structural basis of the different ligand egress mechanisms for fatty acids and small weight compounds. Our investigation provides detailed atomistic insights into the structural features of human CYP2E1-fatty acid complex structures. Furthermore, the ligand-binding characteristics obtained in the present study are helpful for both experimental and computational studies of CYPs and may allow future researchers to achieve desirable changes in enzymatic activities.

Exploring the mechanism how Marburg virus VP35 recognizes and binds dsRNA by molecular dynamics simulations and free energy calculations.

Filoviruses often cause terrible infectious disease which has not been successfully dealt with pharmacologically. All filoviruses encode a unique protein termed VP35 which can mask doubled-stranded RNA to deactivate interferon. The interface of VP35-dsRNA would be a feasible target for structure-based antiviral agent design. To explore the essence of VP35-dsRNA interaction, molecular dynamics simulation combined with MM-GBSA calculations were performed on Marburg virus VP35-dsRNA complex and several mutational complexes. The energetic analysis indicates that nonpolar interactions provide the main driving force for the binding process. Although the intermolecular electrostatic interactions play important roles in VP35-dsRNA interaction, the whole polar interactions are unfavorable for binding which result in a low binding affinity. Compared with wild type VP35, the studied mutants F228A, R271A, and K298A have obviously reduced binding free energies with dsRNA reflecting in the reduction of polar or nonpolar interactions. The results also indicate that the loss of binding affinity for one dsRNA strand would abolish the total binding affinity. Three important residues Arg271, Arg294, and Lys298 which makes the largest contribution for binding in VP35 lose their binding affinity significantly in mutants. The uncovering of VP35-dsRNA recognition mechanism will provide some insights for development of antiviral drug.

[Structure motif guided mining of MHET hydrolase and development of a two-enzyme cascade for plastics depolymerization at mild temperature].

The utilization of polyethylene terephthalate (PET) has caused significant and prolonged ecological repercussions. Enzymatic degradation is an environmentally friendly approach to addressing PET contamination. Hydrolysis of mono(2-hydroxyethyl) terephthalate (MHET), a competitively inhibited intermediate in PET degradation, is catalyzed by MHET degrading enzymes. Herein, we employed bioinformatic methods that combined with sequence and structural information to discover an MHET hydrolase, BurkMHETase. Enzymatic characterization showed that the enzyme was relatively stable at pH 7.5-10.0 and 30-45 ℃. The kinetic parameters kcat and Km on MHET were (24.2±0.5)/s and (1.8±0.2) µmol/L, respectively, which were similar to that of the well-known IsMHETase with higher substrate affinity. BurkMHETase coupled with PET degradation enzymes improved the degradation of PET films. Structural analysis and mutation experiments indicated that BurkMHETase may have evolved specific structural features to hydrolyze MHET. For MHET degrading enzymes, aromatic amino acids at position 495 and the synergistic interactions between active sites or distal amino acids appear to be required for MHET hydrolytic activity. Therefore, BurkMHETase may have substantial potential in a dual-enzyme PET degradation system while the bioinformatic methods can be used to broaden the scope of applicable MHETase enzymes.

Computational redesign of a hydrolase for nearly complete PET depolymerization at industrially relevant high-solids loading.

Biotechnological plastic recycling has emerged as a suitable option for addressing the pollution crisis. A major breakthrough in the biodegradation of poly(ethylene terephthalate) (PET) is achieved by using a LCC variant, which permits 90% conversion at an industrial level. Despite the achievements, its applications have been hampered by the remaining 10% of nonbiodegradable PET. Herein, we address current challenges by employing a computational strategy to engineer a hydrolase from the bacterium HR29. The redesigned variant, TurboPETase, outperforms other well-known PET hydrolases. Nearly complete depolymerization is accomplished in 8 h at a solids loading of 200 g kg-1. Kinetic and structural analysis suggest that the improved performance may be attributed to a more flexible PET-binding groove that facilitates the targeting of more specific attack sites. Collectively, our results constitute a significant advance in understanding and engineering of industrially applicable polyester hydrolases, and provide guidance for further efforts on other polymer types.

Structural and dynamic basis of human cytochrome P450 7B1: a survey of substrate selectivity and major active site access channels.

Cytochrome P450 (CYP) 7B1 is a steroid cytochrome P450 7α-hydroxylase that has been linked directly with bile salt synthesis and hereditary spastic paraplegia type 5 (SPG5). The enzyme provides the primary metabolic route for neurosteroids dehydroepiandrosterone (DHEA), cholesterol derivatives 25-hydroxycholesterol (25-HOChol), and other steroids such as 5α-androstane-3ß,17ß-diol (anediol), and 5α-androstene-3ß,17ß-diol (enediol). A series of investigations including homology modeling, molecular dynamics (MD), and automatic docking, combined with the results of previous experimental site-directed mutagenesis studies and access channels analysis, have identified the structural features relevant to the substrate selectivity of CYP7B1. The results clearly identify the dominant access channels and critical residues responsible for ligand binding. Both binding free energy analysis and total interaction energy analysis are consistent with the experimental conclusion that 25-HOChol is the best substrate. According to 20 ns MD simulations, the Phe cluster residues that lie above the active site, particularly Phe489, are proposed to merge the active site with the adjacent channel to the surface and accommodate substrate binding in a reasonable orientation. The investigation of CYP7B1-substrate binding modes provides detailed insights into the poorly understood structural features of human CYP7B1 at the atomic level, and will be valuable information for drug development and protein engineering.

Exploring the molecular basis of dsRNA recognition by Mss116p using molecular dynamics simulations and free-energy calculations.

DEAD-box proteins are the largest family of helicase that are important in nearly all aspects of RNA metabolism. However, it is unclear how these proteins recognize and bind RNA. Here, we present a detailed analysis of the related DEAD-box protein Mss116p-RNA interaction, using molecular dynamics simulations with MM-GBSA calculations. The energetic analysis indicates that the two strands of double strands RNA (dsRNA) are recognized asymmetrically by Mss116p. The strand 1 of dsRNA provides the main binding affinity. Meanwhile, the nonpolar interaction provides the main driving force for the binding process. Although the contribution of polar interaction is small, it is vital in stabilizing the protein-RNA interaction. Compared with the wild type Mss116p, two studied mutants Q412A and D441A have obviously reduced binding free energies with dsRNA because of the decreasing of polar interaction. Three important residues Lys409, Arg415 and Arg438 lose their binding affinity significantly in mutants. In conclusion, these results complement previous experiments to advance comprehensive understanding of Mss116p-dsRNA interaction. The results also would provide support for the application of similar approaches to the understanding of other DEAD-box protein-RNA complexes.

Molecular dynamic investigations of the mutational effects on structural characteristics and tunnel geometry in CYP17A1.

Cytochrome P450 (CYP) 17A1 is a dual-function monooxygenase with a critical role in the synthesis of many human steroid hormones. The enzyme is an important target for the treatment of breast and prostate cancers that proliferate in response to estrogens and androgens. Despite the ample experimental mutagenesis data, the molecular origin and the structural motifs for the enzymatic activities deficiencies have not been rationalized at the atomic resolution. To this end, we have investigated the effects on structural characteristics and tunnel geometry upon single point mutations in CYP17A1. The MD simulation results combined with PMF calculations and MM-GBSA calculations render an "access mechanism" which encapsulates the effects of mutations on the changes in both structural flexibility and tunnel dynamics, bridging the gap between the theory and the experimentally observed results of enzymatic activity decrease. The underlying molecular mechanism of the heterogeneities in open/closed conformational changes, as well as the wider opening of their respective major tunnels between wt17A1 and two mutants, may be attributed to the closer distances of hydrophobic residues or the disruption of a hydrophobic core. The knowledge of ligand binding characteristics and key residues contributions could guide future experimental and computational work on CYPs so that desirable changes in their enzymatic activities may be achieved. The present study provides important insights into the structure-function relationships of CYP17A1 protein, which could contribute to further understanding about 17-hydroxylase deficiencies and may also improve the understanding of polycystic ovary disease.

Traceless enzymatic protein synthesis without ligation sites constraint.

Protein synthesis and semisynthesis offer immense promise for life sciences and have impacted pharmaceutical innovation. The absence of a generally applicable method for traceless peptide conjugation with a flexible choice of junction sites remains a bottleneck for accessing many important synthetic targets, however. Here we introduce the PALME (protein activation and ligation with multiple enzymes) platform designed for sequence-unconstrained synthesis and modification of biomacromolecules. The upstream activating modules accept and process easily accessible synthetic peptides and recombinant proteins, avoiding the challenges associated with preparation and manipulation of activated peptide substrates. Cooperatively, the downstream coupling module provides comprehensive solutions for sequential peptide condensation, cyclization and protein N/C-terminal or internal functionalization. The practical utility of this methodology is demonstrated by synthesizing a series of bioactive targets ranging from pharmaceutical ingredients to synthetically challenging proteins. The modular PALME platform exhibits unprecedentedly broad accessibility for traceless protein synthesis and functionalization, and holds enormous potential to extend the scope of protein chemistry and synthetic biology.

GRAPE, a greedy accumulated strategy for computational protein engineering.

Nature harbors fascinating enzymatic catalysts with high efficiency, chemo-, regio- and stereoselectivity. However, the insufficient stability of the enzymes often prevents their widespread utilization for industrial processes. Not content with the finite repertoire of naturally occurring enzymes, protein engineering holds promises to extend the applications of the improved enzymes with desired physical and catalytic properties. Herein, we devised a computational strategy (greedy accumulated strategy for protein engineering, GRAPE) to enhance the thermostability of enzymes. Through scanning of all point mutations of the structural and evolutionary consensus analysis, a library containing fewer than 100 mutations was established for characterization. After preliminary experimental verification, effective mutations are clustered in a multidimensional physical property space and then accumulated via the greedy algorithm to produce the final designed enzyme. Using the recently reported IsPETase from Ideonella sakaiensis that decomposes PET under ambient temperatures as a starting point, we adopted the GRAPE strategy to come up with a DuraPETase (TM=77°C, raised by 31°C) which showed drastically enhanced degradation performance (300-fold) on semicrystalline PET films at 40°C.

Characterization and efficient production of a thermostable, halostable and organic solvent-stable cellulase from an oil reservoir.

The manufacture of biofuels from cellulose is regarded as one of practicable strategies to meet increasing energy demand and alleviate environmental issues. Cellulases, which play an important role in the production of second-generation biofuels, are expected to be highly thermostable, halostable and organic solvent-stable to adapt to the harsh conditions in practical application. Here we cloned and characterized a novel cellulase (MaCel) from Mahella australiensis 50-1 BON, an anaerobic thermophile isolated from an oil reservoir. MaCel exhibited excellent thermostability, halostability as well as organic solvent stability, and could be efficiently produced in a yield of 1.7 × 106 U/L in 15 h with inexpensive culture medium. These results indicate that MaCel may be a suitable candidate for industrial applications, illustrating the potential benefits of enzymes from oil reservoir extremophiles in the manufacture of biofuels.

Improving the System Performance of the Asymmetric Biosynthesis of d-Pantoic Acid by Using Artificially Self-Assembled Enzymes in Escherichia coli.

d-Pantoic acid (d-PA) is an important chiral precursor of a broad range of biologically active compounds. The asymmetric synthesis of d-PA through reductase coupling with NADPH regeneration systems is highly promising, but the process is restricted by expensive cofactor consumption and low cofactor recycling frequency. Here, an effective construction of self-assembled ketopantoic acid reductase and glucose dehydrogenase via protein-peptide interaction of PDZ domain and PDZ ligand was established. The self-assembled enzymes exhibited highly ordered two-dimensional threadlike macromolecular structures with improved cofactor regeneration. Furthermore, the bioconversion with whole-cell catalysis showed that the robustness and efficiency of the system with self-assembled enzymes were significantly higher than those of the unassembled enzymes. This study provides a strategy for the effective asymmetric biosynthesis of d-PA with a trace amount of cofactor and shows potential for industrial applications.

Bioretrosynthesis of Functionalized N-Heterocycles from Glucose via One-Pot Tandem Collaborations of Designed Microbes.

The design of multistrain systems has markedly expanded the prospects of using long biosynthetic pathways to produce natural compounds. However, the cooperative use of artificially engineered microbes to synthesize xenobiotic chemicals from renewable carbohydrates is still in its infancy. Here, a microbial system is developed for the production of high-added-value N-heterocycles directly from glucose. Based on a retrosynthetic analysis, eleven genes are selected, systematically modulated, and overexpressed in three Escherichia coli strains to construct an artificial pathway to produce 5-methyl-2-pyrazinecarboxylic acid, a key intermediate in the production of the important pharmaceuticals Glipizide and Acipimox. Via one-pot tandem collaborations, the designed microbes remarkably realize high-level production of 5-methyl-2-pyrazinecarboxylic acid (6.2 ± 0.1 g L-1) and its precursor 2,5-dimethylpyrazine (7.9 ± 0.7 g L-1). This study is the first application of cooperative microbes for the total biosynthesis of functionalized N-heterocycles and provides new insight into integrating bioretrosynthetic principles with synthetic biology to perform complex syntheses.

Reductase of Mutanobactin Synthetase Triggers Sequential C-C Macrocyclization, C-S Bond Formation, and C-C Bond Cleavage.

Mutanobactins (MUBs) and their congeners that contain a macrocycle and/or a thiazepane ring are lipopeptides from Streptococcus mutans, a major causative agent of dental caries. Here we show that the C-terminal reductase domain of MubD releases the lipohexapeptide intermediates in an aldehyde form, which enables a spontaneous C-C macrocyclization. In the presence of a thiol group, the macrocyclized MUBs can further undergo spontaneous C-S bond formation and C-C bond cleavage to generate diverse MUB congeners.

Molecular dynamics investigations of structural and functional changes in Bcl-2 induced by the novel antagonist BDA-366.

Apoptosis is a fundamental biological phenomenon, in which anti- or proapoptotic proteins of the Bcl-2 family regulate a committed step. Overexpression of Bcl-2, the prototypical antiapoptotic protein in this family, is associated with therapy resistance in various human cancers. Accordingly, Bcl-2 inhibitors intended for cancer therapy have been developed, typically against the BH3 domain. Recent experimental evidences have shown that the antiapoptotic function of Bcl-2 is not immutable, and that BDA-366, a novel antagonist of the BH4 domain, converts Bcl-2 from a survival molecule to an inducer of cell death. In this study, the underlying mechanisms of this functional conversion were investigated by accelerated molecular dynamics simulation. Results revealed that Pro127 and Trp30 in the BH4 domain rotate to stabilize BDA-366 via π-π interactions, and trigger a series of significant conformational changes of the α3 helix. This rearrangement blocks the hydrophobic binding site (HBS) in the BH3 domain and further prevents binding of BH3-only proteins, which consequently allows the BH3-only proteins to activate the proapoptotic proteins. Analysis of binding free energy confirmed that BDA-366 cross-inhibits BH3-only proteins, implying negative cooperative effects across separate binding sites. The newly identified blocked conformation of the HBS along with the open to closed transition pathway revealed by this study advances the understanding of the Bcl-2 transition from antiapoptotic to proapoptotic function, and yielded new structural insights for novel drug design against the BH4 domain. Communicated by Ramaswamy H. Sarma.