A multifunctional flavoprotein monooxygenase HspB for hydroxylation and C-C cleavage of 6-hydroxy-3-succinoyl-pyridine.

Flavoprotein monooxygenases catalyze reactions, including hydroxylation and epoxidation, involved in the catabolism, detoxification, and biosynthesis of natural substrates and industrial contaminants. Among them, the 6-hydroxy-3-succinoyl-pyridine (HSP) monooxygenase (HspB) from Pseudomonas putida S16 facilitates the hydroxylation and C-C bond cleavage of the pyridine ring in nicotine. However, the mechanism for biodegradation remains elusive. Here, we refined the crystal structure of HspB and elucidated the detailed mechanism behind the oxidative hydroxylation and C-C cleavage processes. Leveraging structural information about domains for binding the cofactor flavin adenine dinucleotide (FAD) and HSP substrate, we used molecular dynamics simulations and quantum/molecular mechanics calculations to demonstrate that the transfer of an oxygen atom from the reactive FAD peroxide species (C4a-hydroperoxyflavin) to the C3 atom in the HSP substrate constitutes a rate-limiting step, with a calculated reaction barrier of about 20 kcal/mol. Subsequently, the hydrogen atom was rebounded to the FAD cofactor, forming C4a-hydroxyflavin. The residue Cys218 then catalyzed the subsequent hydrolytic process of C-C cleavage. Our findings contribute to a deeper understanding of the versatile functions of flavoproteins in the natural transformation of pyridine and HspB in nicotine degradation.IMPORTANCEPseudomonas putida S16 plays a pivotal role in degrading nicotine, a toxic pyridine derivative that poses significant environmental challenges. This study highlights a key enzyme, HspB (6-hydroxy-3-succinoyl-pyridine monooxygenase), in breaking down nicotine through the pyrrolidine pathway. Utilizing dioxygen and a flavin adenine dinucleotide cofactor, HspB hydroxylates and cleaves the substrate's side chain. Structural analysis of the refined HspB crystal structure, combined with state-of-the-art computations, reveals its distinctive mechanism. The crucial function of Cys218 was never discovered in its homologous enzymes. Our findings not only deepen our understanding of bacterial nicotine degradation but also open avenues for applications in both environmental cleanup and pharmaceutical development.

New modifications of PBAT by a small amount of oxalic acid: Fast crystallization and enhanced degradation in all natural environments.

Biodegradable plastics are often mistakenly thought to be capable of degrading in any environment, but their slow degradation rate in the natural environment is still unsatisfactory. We synthetized a novel series of poly(butylene oxalate-co-adipate-co-terephthalate) (PBOAT) with unchanged melting point (135 °C), high elastic modulus (140 - 219 MPa) and elongation at break (478 - 769%). Fast isothermal crystallization with a semi-crystallization time < 20 s was demonstrated by the PBOAT. In N2 and air atmospheres, the PBOAT maintained the Td,5% higher than 329 °C. They also had good thermal stability at melt processing temperature for more than 20 min. PBOAT exhibited faster hydrolysis and seawater degradation, even under natural soil burial without light, but still kept stable under low humidity conditions during the storage and the shelf-life. Moreover, the hydrolysis mechanisms were clarified based on Fukui function analysis and DFT calculation, indicating that the hydrolysis of PBOAT would be more straightforward. The mechanism of soil burial is also elucidated through detailed characterization of the structure changes. The PBOAT offered a fresh approach to the development of high-performing, naturally degradable materials.

Understanding base and backbone contributions of phosphorothioate DNA for molecular recognition with SBD proteins.

Bacterial DNA phosphorothioate (PT) modification provides a specific anchoring site for sulfur-binding proteins (SBDs). Besides, their recognition patterns include phosphate links and bases neighboring the PT-modified site, thereby bringing about genome sequence-dependent properties in PT-related epigenetics. Here, we analyze the contributions of the DNA backbone (phosphates and deoxyribose) and bases bound with two SBD proteins in Streptomyces pristinaespiralis and coelicolor (SBDSco and SBDSpr). The chalcogen-hydrophobic interactions remained constantly at the anchoring site while the adjacent bases formed conditional and distinctive non-covalent interactions. More importantly, SBD/PT-DNA interactions were not limited within the traditional "4-bp core" range from 5'-I to 3'-III but extended to upstream 5'-II and 5'-III bases and even 5''-I to 5''-III at the non-PT-modified complementary strand. From the epigenetic viewpoint, bases 3'-II, 5''-I, and 5''-III of SBDSpr and 3'-II, 5''-II, and 5''-III of SBDSco present remarkable differentiations in the molecular recognitions. From the protein viewpoint, H102 in SBDSpr and R191 in SBDSco contribute significantly while proline residues at the PT-bound site are strictly conserved for the PT-chalcogen bond. The mutual and make-up mutations are proposed to alter the SBD/PT-DNA recognition pattern, besides additional chiral phosphorothioate modifications on phosphates 5'-II, 5'-II, 3'-I, and 3'-II.

Examining asymmetric pairwise pre-reaction and transition states in enzymatic catalysis by molecular dynamics simulation and quantum mechanics/molecular mechanics calculation.

Here, we present a protocol to examine asymmetric pairwise pre-reaction and transition states in enzymatic catalysis. We describe steps to set up the calculated systems, run umbrella sampling molecular dynamics simulation, and conduct quantum mechanics/molecular mechanics calculations. We also provide analytical scripts to yield potential of mean force of pre-reaction states and reaction barriers. This protocol can generate quantum-mechanistic data for constructing pre-reaction state/transition state machine learning models. For complete details on the use and execution of this protocol, please refer to Luo et al. (2022).1.

Theoretical Studies of Mutual Effects between 6-m-r Hemiketalization and 26-m-r Lactonization in Pimaricin Thioesterase.

Pimaricin is a small polyene macrolide antibiotic and has been broadly used as an antimycotic and antiprotozoal agent in both humans and foods. As a thioesterase in type-I polyketide synthase, pimTE controls the 26-m-r macrolide main chain release in pimaricin biosynthesis. In this work, we sought to determine whether the 6-m-r hemiketal formation was linked to pimTE-catalyzed 26-m-r lactonization. Compared to non-hemiketal TEs, pimTE is characterized by an aspartic acid residue (D179) accessible to the U-turn motif in the acyl-enzyme intermediate. Both the covalent docking and molecular dynamics simulations demonstrate that the reactive conformations for macrocyclic lactonization are drastically promoted by the 6-m-r hemiketal. Moreover, the small-model quantum mechanistic calculations suggest that protic residues can significantly accelerate the 6-m-r hemiketal cyclization. In addition, the post-hemiketal molecular dynamic simulations demonstrate that hydrogen-bonding networks surrounding the substrate U-turn of the hairpin-shaped conformation changes significantly when the 6-m-r hemiketal is formed. In particular, the R-hemiketal intermediate is not only catalyzed by the D179 residue, but also twists the hairpin structure to the 26-m-r lactonizing pre-reaction state. By contrast, the S-hemiketal formation is unlikely catalyzed by D179, which twists the hairpin in an opposite direction. Our results propose that pimTE could be a bi-functional enzyme, which can synergistically catalyze tandem 6-m-r and 26-m-r formations during the main-chain release of pimaricin biosynthesis.

Visually induced γ band rhythm in spatial summation beyond the receptive field in mouse primary visual cortex.

Recent studies in many kinds of mammals have established the existence of multiple γ rhythms in the cerebral cortex subserving different functions. In the primary visual cortex (V1), visually induced γ rhythms are dependent on stimulus features. However, experimental findings of γ power induced by varying the size of the drifting grating are inconsistent. Here, we reinvestigated the spatial summation properties of visually induced spike and γ rhythm activities in mouse V1. Our results show that drifting sinusoidal grating stimuli mainly induce 2 γ band rhythms, including a low-frequency band (25-45 Hz) and a high-frequency band (55-75 Hz). Unlike previous findings, we discovered that visually induced γ power could also exhibit extrareceptive field (ERF) modulatory properties. The modulation by ERF stimulation could be either suppressive, countersuppressive, or nonsuppressive, mostly similar to the local spike activity. Moreover, further analysis of the neuron group exhibiting surround suppression in both spike and γ activity revealed that the strength of the surround suppression and the receptive field size showed moderate correlations between measurements by spike and γ rhythm activity. Our findings improve the understanding of the characteristics and neural mechanisms of induced γ rhythms in visual spatial summation.

Chemical control over the conversion between bicyclic and polycyclic terpenes by fungal bifunctional terpene synthases.

Fungal bifunctional terpene synthases (BFTSs) reportedly associate with a series of new skeletons of di/sesterterpenes. However, the molecular mechanisms underlying the variabilities in the ring system of BFTS-catalyzed products are not well understood. In this study, we identified a key site, S89/L89, that controls the conversion between bicyclic and polycyclic terpene skeletons catalyzed by two BFTSs, BsPS and FoFS. Our analysis revealed that a mutation on site 89 in the BFTSs alters the carbocation transportation pathway and redirects the competing reactions for previously unreported terpenes.

Origin of iodine preferential attack at sulfur in phosphorothioate and subsequent P-O or P-S bond dissociation.

Iodine-induced cleavage at phosphorothioate DNA (PT-DNA) is characterized by extremely high sensitivity (∼1 phosphorothioate link per 106 nucleotides), which has been used for detecting and sequencing PT-DNA in bacteria. Despite its foreseeable potential for wide applications, the cleavage mechanism at the PT-modified site has not been well established, and it remains unknown as to whether or not cleavage of the bridging P-O occurs at every PT-modified site. In this work, we conducted accurate ωB97X-D calculations and high-performance liquid chromatography-mass spectrometry to investigate the process of PT-DNA cleavage at the atomic and molecular levels. We have found that iodine chemoselectively binds to the sulfur atom of the phosphorothioate link via a strong halogen-chalcogen interaction (a type of halogen bond, with binding affinity as high as 14.9 kcal/mol) and thus triggers P-O bond cleavage via phosphotriester-like hydrolysis. Additionally, aside from cleavage of the bridging P-O bond, the downstream hydrolyses lead to unwanted P-S/P-O conversions and a loss of the phosphorothioate handle. The mechanism we outline helps to explain specific selectivity at the PT-modified site but also predicts the dynamic stoichiometry of P-S and P-O bond breaking. For instance, Tris is involved in the cascade derivation of S-iodo-phosphorothioate to S-amino-phosphorothioate, suppressing the S-iodo-phosphorothioate hydrolysis to a phosphate diester. However, hydrolysis of one-third of the Tris-O-grafting phosphotriester results in unwanted P-S/P-O conversions. Our study suggests that bacterial DNA phosphorothioation may more frequently occur than previous bioinformatic estimations have predicted from iodine-induced deep sequencing data.

Molecular recognition between bacterial phosphorothioate DNA and sulfur-binding domain (SBD): competition between the water cage and chalcogen-hydrophobic packet.

Bacterial DNA phosphorothioation (PT) physiologically and stereo-specifically replaces a non-bridging oxygen in a phosphate link with a sulfur atom, which can be recognized by a highly conserved sulfur-binding domain (SBD). Here we conducted thermodynamic integration (TI), molecular dynamics simulation, and quantum chemical calculations to decipher the specific molecular interactions between PT-DNA and SBD in Streptomyces coelicolor type IV restriction enzyme ScoMcrA. The TI-calculated binding affinity of (5'-CCGRp-PSGCCGG-3')2 is larger than that of (5'-CCGGCCGG-3')2 by about 7.4-7.7 kcal mol-1. The binding difference dominantly stems from hydration energy of non-phosphorothioate DNA (9.8-10.6 kcal mol-1) in aqueous solution, despite the persistent preference of 2.6-3.2 kcal mol-1 in the DNA-SBD MD simulations. Furthermore, the quantum chemical calculations reveal an unusual non-covalent interaction in the phosphorothioate-binding scenario, where the PS⋯NP165 chalcogen bond prevails the PS⋯HCß vdW interactions from the adjacent residues H116-R117-Y164-P165-A168. Thus, the chalcogen-hydrophobic interaction pulls PT-DNA into the SBD binding pocket while the water cage pulls a normal DNA molecule out. The synergetic mechanism suggests the special roles of the proline pyrrolidine group in the SBD proteins, consistent with the experimental observations in the X-ray crystallography and structural bioinformatics analysis.

Acid-triggered, degradable and high strength-toughness copolyesters: Comprehensive experimental and theoretical study.

The popularization and widespread use of degradable polymers is hindered by their poor mechanical properties. It is of great importance to find a balance between degradation and mechanical properties. Herein, poly(butylene terephthalate) (PBT) modified by SPG diol from 10% to 40 mol% were synthesized through a two-step polycondensation reaction. Chemical structures, thermal properties, mechanical properties, viscoelastic behavior and degradation of poly(butylene terephthalate-co-spirocyclic terephthalate) (PBST) were investigated. The SPG could toughen the copolyesters and the elongation at break of PBST20 was up to 260%. Moreover, the introduction of SPG enables to provide an acid-triggered degradable unit in the main chain. PBSTs copolymers maintain stable structures in a neutral environment, and the degradation under acid conditions will be unlocked. As tailoring the content of SPG, the degradation rate of the chain scission in response to acid stimuli will be adjusted. The acid degradation was proved to be occurred at the SPG units in the amorphous phase by DSC, XRD, GPC and 1H NMR tests. After the acid degradation, the hydrolysis rate will also be accelerated, adapting to the requirements of different degradation schedules. The plausible hydrolytic pathways and mechanisms were proposed based on Fukui function analysis and density functional theory (DFT) calculation.

Design of 2,5-furandicarboxylic based polyesters degraded in different environmental conditions: Comprehensive experimental and theoretical study.

Nowadays, the promotion and application of aliphatic-aromatic copolyesters, such as poly (butylene adipate-co-terephthalate) (PBAT), are growing into a general trend. Although the structures of diacids exerted substantial impacts on degradation behavior, the underlying mechanisms have rarely been studied. In this work, 2,5-Furandicarboxylic acid was combined with succinic acid (PBSF), adipic acid (PBAF) and diglycolic acid (PBDF) to prepare three kinds of copolyesters. They showed unique degradation behaviors in buffer, enzyme environment and artificial seawater. These characteristics are closely related to the structural compositions of diacids. PBAFs displayed impressive biodegradability when catalyzed by Candida antarctica lipase B (CALB), while the more hydrophilic PBDFs exhibited faster hydrolysis in both buffer and artificial seawater. PBSFs, with hydrophobic and short segments, obtained a relatively slower rate of hydrolysis and enzymatic degradation. The reactivity sites and hydrolytic pathway were revealed by the combination of DFT calculation and Fukui function analysis. MD simulations, QM/MM optimizations and theozyme calculations showed that PBAF-CALB was prone to form a pre-reaction state, leading to the reduced energy barrier in the acylation process. This work revealed the effects of different structural features of diacids on polymer degradation and paved a way to design target biodegradable polymers in different degradation conditions.

Exploring the Molecular Basis of Substrate and Product Selectivities of Nocardicin Bifunctional Thioesterase.

D-amino acid introduction in peptides can enrich their biological activities and pharmacological properties as potential drugs. This achievement of stereochemical inversion usually owes to an epimerase or racemase. Interestingly, a unique bifunctional thioesterase (NocTE), which is incorporated in nonribosomal peptide synthetase (NRPS) NocA-NocB assembly line for the biosynthesis of monocyclic ß-lactam antibiotic nocardicin A, can control the generation of D-products with high stereochemical purity. However, the molecular basis of NocTE selectivity on substrates and products is still unclear. Herein, we constructed a series of systems with different peptides varying in stereochemistry, length, and composition to investigate the substrate selectivity. The studies on binding affinities and loading conformations elucidated the important roles of peptide length and ß-lactam ring in substrate selectivity. Through energy decomposition and interaction analyses, some key residues involved in substrate selectivity were captured. On the other hand, natural product undergoing epimerization was found to be liberated from the active pocket more easily in comparison with its diastereomer (epi-nocardicin G), explaining the superiority of nocardicin G. These results provide detailed molecular insights into the exquisite control of substrate and product scopes for NocTE, and encourage to diversification of substrates and final products for NRPS assembly line. The molecular insights into substrate and product selectivities of unique bifunctional thioesterase NocTE were illustrated via several molecular simulations and free energy calculations, contributing to expanding substrate and product scopes of nonribosomal peptide synthetases.

Computer-aided understanding and engineering of enzymatic selectivity.

Enzymes offering chemo-, regio-, and stereoselectivity enable the asymmetric synthesis of high-value chiral molecules. Unfortunately, the drawback that naturally occurring enzymes are often inefficient or have undesired selectivity toward non-native substrates hinders the broadening of biocatalytic applications. To match the demands of specific selectivity in asymmetric synthesis, biochemists have implemented various computer-aided strategies in understanding and engineering enzymatic selectivity, diversifying the available repository of artificial enzymes. Here, given that the entire asymmetric catalytic cycle, involving precise interactions within the active pocket and substrate transport in the enzyme channel, could affect the enzymatic efficiency and selectivity, we presented a comprehensive overview of the computer-aided workflow for enzymatic selectivity. This review includes a mechanistic understanding of enzymatic selectivity based on quantum mechanical calculations, rational design of enzymatic selectivity guided by enzyme-substrate interactions, and enzymatic selectivity regulation via enzyme channel engineering. Finally, we discussed the computational paradigm for designing enzyme selectivity in silico to facilitate the advancement of asymmetric biosynthesis.

Reductive inactivation of the hemiaminal pharmacophore for resistance against tetrahydroisoquinoline antibiotics.

Antibiotic resistance is becoming one of the major crises, among which hydrolysis reaction is widely employed by bacteria to destroy the reactive pharmacophore. Correspondingly, antibiotic producer has canonically co-evolved this approach with the biosynthetic capability for self-resistance. Here we discover a self-defense strategy featuring with reductive inactivation of hemiaminal pharmacophore by short-chain dehydrogenases/reductases (SDRs) NapW and homW, which are integrated with the naphthyridinomycin biosynthetic pathway. We determine the crystal structure of NapW·NADPH complex and propose a catalytic mechanism by molecular dynamics simulation analysis. Additionally, a similar detoxification strategy is identified in the biosynthesis of saframycin A, another member of tetrahydroisoquinoline (THIQ) antibiotics. Remarkably, similar SDRs are widely spread in bacteria and able to inactive other THIQ members including the clinical anticancer drug, ET-743. These findings not only fill in the missing intracellular events of temporal-spatial shielding mode for cryptic self-resistance during THIQs biosynthesis, but also exhibit a sophisticated damage-control in secondary metabolism and general immunity toward this family of antibiotics.

Phosphorothioate-DNA bacterial diet reduces the ROS levels in C. elegans while improving locomotion and longevity.

DNA phosphorothioation (PT) is widely distributed in the human gut microbiome. In this work, PT-diet effect on nematodes was studied with PT-bioengineering bacteria. We found that the ROS level decreased by about 20-50% and the age-related lipofuscin accumulation was reduced by 15-25%. Moreover, the PT-feeding worms were more active at all life periods, and more resistant to acute stressors. Intriguingly, their lifespans were prolonged by ~21.7%. Comparative RNA-seq analysis indicated that many gene expressions were dramatically regulated by PT-diet, such as cysteine-rich protein (scl-11/12/13), sulfur-related enzyme (cpr-2), longevity gene (jnk-1) and stress response (sod-3/5, gps-5/6, gst-18/20, hsp-12.8). Both the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis suggested that neuroactivity pathways were upregulated, while phosphoryl transfer and DNA-repair pathways were down-regulated in good-appetite young worms. The findings pave the way for pro-longevity of multicellular organisms by PT-bacterial interference.

Evolutionary coupling-inspired engineering of alcohol dehydrogenase reveals the influence of distant sites on its catalytic efficiency for stereospecific synthesis of chiral alcohols.

Alcohol dehydrogenase (ADH) has attracted much attention due to its ability to catalyze the synthesis of important chiral alcohol pharmaceutical intermediates with high stereoselectivity. ADH protein engineering efforts have generally focused on reshaping the substrate-binding pocket. However, distant sites outside the pocket may also affect its activity, although the underlying molecular mechanism remains unclear. The current study aimed to apply evolutionary coupling-inspired engineering to the ADH CpRCR and to identify potential mutation sites. Through conservative analysis, phylogenic analysis and residues distribution analysis, the co-evolution hotspots Leu34 and Leu137 were confirmed to be highly evolved under the pressure of natural selection and to be possibly related to the catalytic function of the protein. Hence, Leu34 and Leu137, far away from the active center, were selected for mutation. The generated CpRCR-L34A and CpRCR-L137V variants showed high stereoselectivity and 1.24-7.81 fold increase in k cat /K m value compared with that of the wild type, when reacted with 8 aromatic ketones or ß-ketoesters. Corresponding computational study implied that L34 and L137 may extend allosteric fluctuation in the protein structure from the distal mutational site to the active site. Moreover, the L34 and L137 mutations modified the pre-reaction state in multiple ways, in terms of position of the hydride with respect to the target carbonyl. These findings provide insights into the catalytic mechanism of the enzyme and facilitate its regulation from the perspective of the site interaction network.

Natural Syringyl Mediators Accelerate Laccase-Catalyzed ß-O-4 Cleavage and Cα-Oxidation of a Guaiacyl Model Substrate via an Aggregation Mechanism.

Laccase-mediator systems (LMSs) have been intensively investigated in lignin degradation. Although only natural metabolites are available for fungal lignin degradation, mediator molecules from metabolites have received substantially less attention than artificial organic-synthetic compounds. It remains unclear which metabolites can accelerate laccase-catalyzed reactions and how those natural mediators influence lignin degradation. In this work, we evaluated Trametes versicolor laccase-catalyzed reaction kinetics on a lignin guaiacyl subunit model (guaiacylglycerol-ß-guaiacyl ether, G-ß-GE) in the presence of a group of lignin syringyl subunit molecules: syringaldehyde, acetosyringone, and methyl syringate. We then compare their performance to a well-known synthetic mediator ABTS, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid). Time-resolved UPLC-TOF-MS revealed that the syringyl mediators were more effective in accelerating the ß-O-4 cleavage and Cα-oxidation of G-ß-GE than ABTS under laccase-catalysis, despite the syringyl compounds possessing slower individual oxidation rates. In addition, the product profile of polymerization was also promoted dramatically, compared to that of the ABTS/laccase system. The LMS kinetic modeling suggested that mediator-substrate aggregation played a critical role in the laccase-mediator system; in which, the lignin syringyl and guaiacyl subunits likely form a π-π stacking van der Waals complex that can be oxidized faster than the syringyl or guaiacyl monomers by themselves. This syringyl-guaiacyl aggregation hypothesis postulates that the weak interactions in lignin biopolymers are able to accelerate the laccase-catalyzed biodegradation.

Biosynthesis of para-Cyclophane-Containing Hirsutellone Family of Fungal Natural Products.

Hirsutellones are fungal natural products containing a macrocyclic para-cyclophane connected to a decahydrofluorene ring system. We have elucidated the biosynthetic pathway for pyrrocidine B (3) and GKK1032 A2 (4). Two small hypothetical proteins, an oxidoreductase and a lipocalin-like protein, function cooperatively in the oxidative cyclization of the cyclophane, while an additional hypothetical protein in the pyrrocidine pathway catalyzes the exo-specific cycloaddition to form the cis-fused decahydrofluorene.