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.

High-Molecular-Weight and Light-Colored Disulfide-Bond-Embedded Polyesters: Accelerated Hydrolysis Triggered by Redox Responsiveness.

Disulfide bonds have attracted considerable attention due to their reduction responsiveness, but it is crucial and challenging to prepare disulfide-bond-based polyesters by melt polycondensation. Herein, the inherently poor thermal stability of the S-S bond in melting polycondensation was overcome. Moreover, poly(butylene succinate-co-dithiodipropionate) (PBSDi) with a light color and high molecular weights (Mn values up to 84.7 kg/mol) was obtained. These polyesters can be applied via melt processing with Td,5% > 318 °C. PBSDi10-PBSDi40 shows good crystallizability (crystallinity 56-38%) and compact lamellar thickness (2.9-3.2 nm). Compared with commercial poly(butylene adipate-co-terephthalate) (PBAT), the elevated mechanical and barrier performances of PBSDi make them better packaging materials. For the degradation behavior, the disulfide monomer obviously accelerates the enzyme degradation but has a weaker effect on hydrolysis. In 0.1 mol/L or higher concentrations of H2O2 solutions, the oxidation of disulfide bonds to sulfoxide and sulfone groups can be realized. This process results in a stronger nucleophilic attack, as confirmed by the Fukui function and DFT calculations. Additionally, the greater polarity and hydrophilicity of oxidation products, proved by noncovalent interaction analysis, accelerate the hydrolysis of polyesters. Moreover, glutathione-responsive breakage, from polymers to oligomers, is confirmed by an accelerated decline in molecular weight. Our research offers fresh perspectives on the effective synthesis of the disulfide polyester and lays a solid basis for the creation of high-performance biodegradable polyesters that degrade on demand.

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.

Characterization of a novel aromatic ring-hydroxylating oxygenase, NarA2B2, from thermophilic Hydrogenibacillus sp. strain N12.

Polycyclic aromatic hydrocarbons (PAHs) are harmful to human health due to their carcinogenic, teratogenic, and mutagenic effects. A thermophilic Hydrogenibacillus sp. strain N12 capable of degrading a variety of PAHs and derivatives was previously isolated. In this study, an aromatic ring-hydroxylating oxygenase, NarA2B2, was identified from strain N12, with substrate specificity including naphthalene, phenanthrene, dibenzothiophene, fluorene, acenaphthene, carbazole, biphenyl, and pyrene. NarA2B2 was proposed to add one or two atoms of molecular oxygen to the substrate and catalyze biphenyl at C-2, 2 or C-3, 4 positions with different characteristics than before. The key catalytic amino acids, H222, H227, and D379, were identified as playing a pivotal role in the formation of the 2-his-1-carboxylate facial triad. Furthermore, we conducted molecular docking and molecular dynamics simulations, notably, D219 enhanced the stability of the iron center by forming two stable hydrogen bonds with H222, while the mutation of F216, T223, and H302 modulated the catalytic activity by altering the pocket's size and shape. Compared to the wild-type (WT) enzyme, the degradation ratios of acenaphthene by F216A, T223A, and H302A had an improvement of 23.08%, 26.87%, and 29.52%, the degradation ratios of naphthalene by T223A and H302A had an improvement of 51.30% and 65.17%, while the degradation ratio of biphenyl by V236A had an improvement of 77.94%. The purified NarA2B2 was oxygen-sensitive when it was incubated with L-ascorbic acid in an anaerobic environment, and its catalytic activity was restored in vitro. These results contribute to a better understanding of the molecular mechanism responsible for PAHs' degradation in thermophilic microorganisms.IMPORTANCE(i) A novel aromatic ring-hydroxylating oxygenase named NarA2B2, capable of degrading multiple polycyclic aromatic hydrocarbons and derivatives, was identified from the thermophilic microorganism Hydrogenibacillus sp. N12. (ii) The degradation characteristics of NarA2B2 were characterized by adding one or two atoms of molecular oxygen to the substrate. Unlike the previous study, NarA2B2 catalyzed biphenyl at C-2, 2 or C-3, 4 positions. (iii) Catalytic sites of NarA2B2 were conserved, and key amino acids F216, D219, H222, T223, H227, V236, F243, Y300, H302, W316, F369, and D379 played pivotal roles in catalysis, as confirmed by protein structure prediction, molecular docking, molecular dynamics simulations, and point mutation.

An Intelligent Synthetic Bacterium for Chronological Toxicant Detection, Biodegradation, and Its Subsequent Suicide.

Modules, toolboxes, and synthetic biology systems may be designed to address environmental bioremediation. However, weak and decentralized functional modules require complex control. To address this issue, an integrated system for toxicant detection and biodegradation, and subsequent suicide in chronological order without exogenous inducers is constructed. Salicylic acid, a typical pollutant in industrial wastewater, is selected as an example to demonstrate this design. Biosensors are optimized by regulating the expression of receptors and reporters to get 2-fold sensitivity and 6-fold maximum output. Several stationary phase promoters are compared, and promoter Pfic is chosen to express the degradation enzyme. Two concepts for suicide circuits are developed, with the toxin/antitoxin circuit showing potent lethality. The three modules are coupled in a stepwise manner. Detection and biodegradation, and suicide are sequentially completed with partial attenuation compared to pre-integration, except for biodegradation, being improved by the replacements of ribosome binding site. Finally, a long-term stability test reveals that the engineered strain maintained its function for ten generations. The study provides a novel concept for integrating and controlling functional modules that can accelerate the transition of synthetic biology from conceptual to practical applications.

Glycolipotoxicity conferred tendinopathy through ferroptosis dictation of tendon-derived stem cells by YAP activation.

Tendinopathy is a condition characterized by chronic, complex, and multidimensional pathological changes in the tendons. The etiology of tendinopathy is the combination of several factors, and diabetes mellitus (DM) is a risk factor. Increasing evidence has shown that the diabetic microenvironment plays an important role in tendinopathy. However, the mechanism causing tendinopathy in patients with DM remains unclear. Our study found that ferroptosis played an important role in tendinopathy in patients with DM. In vitro, high glucose and high fat treatment was used to simulate the DM microenvironment. Results showed that such a mechanism significantly increased ferroptosis, which was characterized by mass cell death, lipid peroxide accumulation, mitochondrial morphological changes, mitochondrial membrane potential decline, iron overload, and the activation of ferroptosis-related genes, in tendon-derived stem cells cultured in vitro. In the animal studies, db/db mice were used in the DM model, and the db mice had severe tendon injury and high ACSL4 and TfR1 expressions. These phenomena could be alleviated by the ferroptosis inhibitor ferrostatin-1. In conclusion, ferroptosis is associated with tendinopathy in patients with DM, and ferroptosis targeting may be a novel approach for treating diabetic tendinopathy. Our results can provide a new strategy for managing tendinopathy clinically in patients with DM.

The role of Gel-Ppy-modified nerve conduit on the repair of sciatic nerve defect in rat model.

The serious clinical challenge of peripheral nerve injury (PNI) is nerve regeneration. Nerve conduit represents a promising strategy to contribute to nerve regeneration by bridging injured nerve gaps. However, due to a unique microenvironment of nerve tissue, autologous nerves have not been substituted by nerve conduit. Nerve regeneration after nerve conduit implantation depends on many factors, such as conductivity and biocompatibility. Therefore, Gelatin (Gel) with biocompatibility and polypyrrole (Ppy) with conductivity is highly concerned. In this paper, Gel-Ppy modified nerve conduit was fabricated with great biocompatibility and conductivity to evaluate its properties of enhancing nerve regeneration in vivo and in vitro. The proliferation of Schwann cells on Gel-Ppy modified nerve conduit was remarkably increased. Consistent with in vitro results, the Gel-Ppy nerve conduit could contribute to the regeneration of Schwann cell in vivo. The axon diameters and myelin sheath thickness were also enhanced, resulting in the amelioration of muscle atrophy, nerve conduction, and motor function recovery. To explain this interesting phenomenon, western blot results indicated that the Gel-Ppy conduit facilitated nerve regeneration via upregulating the Rap1 pathway to induce neurite outgrowth. Therefore, the above results demonstrated that Gel-Ppy modified nerve conduit could provide an acceptable microenvironment for nerve regeneration and be popularized as a novel therapeutic strategy of PNI.

Insulin-like growth factor binding protein 4 loaded electrospun membrane ameliorating tendon injury by promoting retention of IGF-1.

Tendon injury is one of the most common musculoskeletal disorders that impair joint mobility and lower quality of life. The limited regenerative capacity of tendon remains a clinical challenge. Local delivery of bioactive protein is a viable therapeutic approach for tendon healing. Insulin-like growth factor binding protein 4 (IGFBP-4) is a secreted protein capable of binding and stabilizing insulin-like growth factor 1 (IGF-1). Here, we applied an aqueous-aqueous freezing-induced phase separation technology to obtain the IGFBP4-encapsulated dextran particles. Then, we added the particles into poly (L-lactic acid) (PLLA) solution to fabricate IGFBP4-PLLA electrospun membrane for efficient IGFBP-4 delivery. The scaffold showed excellent cytocompatibility and a sustained release of IGFBP-4 for nearly 30 days. In cellular experiments, IGFBP-4 promoted tendon-related and proliferative markers expression. In a rat Achilles tendon injury model, immunohistochemistry and quantitative real-time polymerase chain reaction confirmed better outcomes by using the IGFBP4-PLLA electrospun membrane at the molecular level. Furthermore, the scaffold effectively promoted tendon healing in functional performance, ultrastructure and biomechanical properties. We found addition of IGFBP-4 promoted IGF-1 retention in tendon postoperatively and then facilitated protein synthesis via IGF-1/AKT signaling pathway. Overall, our IGFBP4-PLLA electrospun membrane provides a promising therapeutic strategy for tendon injury.

Heterologous expression of family GH11 Aspergillus niger xylanase B (AnXylB11) in Pichia pastoris and competitive inhibition by riceXIP: An experimental and simulation study.

The family GH11 Aspergillus niger JL15 xylanase B (AnXylB11) was heterologously expressed in Pichia pastoris X33. The recombinant AnXylB11 (reAnXylB11) was secreted into the culture medium with a molecular weight of approximately 33.0 kDa. The optimal temperature and pH of reAnXylB11 were 40 â„ƒ and 5.0, respectively. reAnXylB11 released xylobiose (X2)-xylohexaose (X6) from beechwood xylan, with xylotriose (X3) as the primary product. The hydrolysates showed significant antioxidant activity. reAnXylB11 was also competitively inhibited by recombinant rice xylanase inhibitory protein (rePriceXIP), with an inhibition constant (Ki) of 106.9 nM. Molecular dynamics (MD) simulations, non-covalent interactions (NCI), and binding free energy calculation and decomposition were conducted to decipher the interactional features between riceXIP and AnXyB11. Representative conformation of riceXIP-AnXylB11 complex showed that a U-shaped long loop between α4 and ß5 (K143-L152) of riceXIP was protruded into the catalytic groove and formed tight interaction with many key residues of AnXylB11. The binding free energy of riceXIP-AnXylB11 was calculated to be - 46.1 ± 10.5 kcal/mol, with Coulomb and van der Waals forces contributing the most. NCI analysis showed that the hydrogen bonding networks such as R148riceXIP-E98AnXyl11B, K143riceXIP-D138AnXyl11B and R148riceXIP-E189AnXyl11B provided terrific contributions to the interface interaction. The Laplacian of electron density values of atom pairs R148riceXIP@ 2HH1-E89AnXylB11@OE2 and N142riceXIP@ 1HD2-D138AnXylB11@OD1 were 0.12190 and 0.16009 a.u., respectively. Exploring the interactional features between xylanase and inhibitor protein may aid in constructing mutant xylanase that is insensitive to xylanase inhibitory proteins (XIs).

Exploring competitive inhibition of a family 10 xylanase derived from Hu sheep rumen microbiota by Oryza sativa xylanase inhibitor protein: In vitro and in silico perspectives.

The catalytic domain of family GH10 xylanase, XYN-LXY_CD derived from Hu sheep rumen microbiota was expressed in Pichia pastoris X33. The special activity of reXYN-LXY_CD in the culture supernatant was 232.56 U/mg. The optima of reXYN-LXY_CD were 53 °C and pH 7.0. Recombinant Oryza sativa xylanase inhibitor protein (rePOsXIP) competitively inhibited reXYN-LXY_CD with an inhibition constant (Ki) value of 237.37 nM. The concentration of hydrolysates released from beechwood xylan by reXYN-LXY_CD reduced when rePOsXIP was added into the hydrolytic system. Fluorescence of reXYN-LXY_CD was statically quenched by rePOsXIP in a dose-dependent manner. The details in intermolecular interaction between XYN-LXY_CD and OsXIP were investigated by using molecular dynamics (MD) simulations, binding free energy computation and non-covalent interactions (NCI) analysis. Hydrogen bonding and van der Waals played indispensable roles in the XYN-LXY_CD/OsXIP interaction. The α-7 helix of OsXIP tightly occupied the catalytic pocket of XYN-LXY_CD with hydrogen bonding such as K239OsXIP-N261/Q292/E197XYN-LXY_CD (E197, the acid-base catalytic residue), D236OsXIP-K327XYN-LXY_CD and Q242OsXIP-E211/Q212XYN-LXY_CD. Based on the quantum theory of atoms in molecules (QTAIM), the Laplacian of electron density and core-valence bifurcation index of HZ3K239-OE2E197 were 0.1025 a.u. and 0.002218, respectively. Elucidating the mechanism underlying xylanase-inhibitor interactions might help construct XYN-LXY_CD mutants that gain resistance to XIPs and high catalytic activity, which would be more efficient in feed additives in livestock.

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.

MicroRNA engineered umbilical cord stem cell-derived exosomes direct tendon regeneration by mTOR signaling.

BACKGROUND: Exosomes are extracellular vesicles of nano-structures and represent an emerging nano-scale acellular therapy in recent years. Tendon regeneration is a sophisticated process in the field of microsurgery due to its poor natural healing ability. To date, no successful long-term solution has been provided for the healing of tendon injuries. Functional recovery requires advanced treatment strategies. Human umbilical cord mesenchymal stem cell-derived exosomes (HUMSC-Exos) are considered as promising cell-free therapeutic agents. However, few studies reported their potential in the tendon repair previously. In this study, we explored the roles and underlying mechanisms of HUMSC-Exos in the tendon regeneration. RESULTS: Expression of tendon-specific markers in, and collagen deposition by, tendon-derived stem cells (TDSCs) treated with HUMSC-Exos increased in vitro. In a rat Achilles tendon injury model, treatment with HUMSC-Exos improved the histological structure, enhanced tendon-specific matrix components, and optimized biomechanical properties of the Achilles tendon. Findings in miRNA sequencing indicated a significant increase in miR-29a-3p in HUMSC-Exo-treated Achilles tendons. Next, luciferase assay in combination with western blot identified phosphatase and tensin homolog (PTEN) as the specific target of miR-29a-3p. Furthermore, we applied a miR-29a-3p-specific agonist to engineer HUMSC-Exos. These HUMSC-Exos overexpressing miR-29a-3p amplified the gain effects of HUMSC-Exos on tendon healing in vivo. To explore the underlying mechanisms, a transforming growth factor-ß1 (TGF-ß1) inhibitor (SB-431542), mTOR inhibitor (rapamycin), and engineered HUMSC-Exos were employed. The results showed that TGF-ß1 and mTOR signaling were involved in the beneficial effects of HUMSC-Exos on tendon regeneration. CONCLUSION: The findings in our study suggest that PTEN/mTOR/TGF-ß1 signaling cascades may be a potential pathway for HUMSC-Exos to deliver miR-29a-3p for tendon healing and implicate a novel therapeutic strategy for tendon regeneration via engineered stem cell-derived exosomes.

Genetic mapping of highly versatile and solvent-tolerant Pseudomonas putida B6-2 (ATCC BAA-2545) as a 'superstar' for mineralization of PAHs and dioxin-like compounds.

Polycyclic aromatic hydrocarbons (PAHs) and dioxin-like compounds, including sulfur, nitrogen and oxygen heterocycles, are widespread and toxic environmental pollutants. A wide variety of microorganisms capable of growing with aromatic polycyclic compounds are essential for bioremediation of the contaminated sites and the Earth's carbon cycle. Here, cells of Pseudomonas putida B6-2 (ATCC BAA-2545) grown in the presence of biphenyl (BP) are able to simultaneously degrade PAHs and their derivatives, even when they are present as mixtures, and tolerate high concentrations of extremely toxic solvents. Genetic analysis of the 6.37 Mb genome of strain B6-2 reveals coexistence of gene clusters responsible for central catabolic systems of aromatic compounds and for solvent tolerance. We used functional transcriptomics and proteomics to identify the candidate genes associated with catabolism of BP and a mixture of BP, dibenzofuran, dibenzothiophene and carbazole. Moreover, we observed dynamic changes in transcriptional levels with BP, including in metabolic pathways of aromatic compounds, chemotaxis, efflux pumps and transporters potentially involved in adaptation to PAHs. This study on the highly versatile activities of strain B6-2 suggests it to be a potentially useful model for bioremediation of polluted sites and for investigation of biochemical, genetic and evolutionary aspects of Pseudomonas.

Structure-guided insights into heterocyclic ring-cleavage catalysis of the non-heme Fe (II) dioxygenase NicX.

Biodegradation of aromatic and heterocyclic compounds requires an oxidative ring cleavage enzymatic step. Extensive biochemical research has yielded mechanistic insights about catabolism of aromatic substrates; yet much less is known about the reaction mechanisms underlying the cleavage of heterocyclic compounds such as pyridine-ring-containing ones like 2,5-hydroxy-pyridine (DHP). 2,5-Dihydroxypyridine dioxygenase (NicX) from Pseudomonas putida KT2440 uses a mononuclear nonheme Fe(II) to catalyze the oxidative pyridine ring cleavage reaction by transforming DHP into N-formylmaleamic acid (NFM). Herein, we report a crystal structure for the resting form of NicX, as well as a complex structure wherein DHP and NFM are trapped in different subunits. The resting state structure displays an octahedral coordination for Fe(II) with two histidine residues (His265 and His318), a serine residue (Ser302), a carboxylate ligand (Asp320), and two water molecules. DHP does not bind as a ligand to Fe(II), yet its interactions with Leu104 and His105 function to guide and stabilize the substrate to the appropriate position to initiate the reaction. Additionally, combined structural and computational analyses lend support to an apical dioxygen catalytic mechanism. Our study thus deepens understanding of non-heme Fe(II) dioxygenases.

Genetic Interference Analysis Reveals that Both 3-Hydroxybenzoic Acid and 4-Hydroxybenzoic Acid Are Involved in Xanthomonadin Biosynthesis in the Phytopathogen Xanthomonas campestris pv. campestris.

A characteristic feature of phytopathogenic Xanthomonas bacteria is the production of yellow membrane-bound pigments called xanthomonadins. Previous studies showed that 3-hydroxybenzoic acid (3-HBA) was a xanthomonadin biosynthetic intermediate and also, that it had a signaling role. The question of whether the structural isomers 4-HBA and 2-HBA (salicylic acid) have any role in xanthomonadin biosynthesis remained unclear. In this study, we have selectively eliminated 3-HBA, 4-HBA, or the production of both by expression of the mhb, pobA, and pchAB gene clusters in the Xanthomonas campestris pv. campestris strain XC1. The resulting strains were different in pigmentation, virulence factor production, and virulence. These results suggest that both 3-HBA and 4-HBA are involved in xanthomonadin biosynthesis. When both 3-HBA and 4-HBA are present, X. campestris pv. campestris prefers 3-HBA for Xanthomonadin-A biosynthesis; the 3-HBA-derived Xanthomonadin-A was predominant over the 4-HBA-derived xanthomonadin in the wild-type strain XC1. If 3-HBA is not present, then 4-HBA is used for biosynthesis of a structurally uncharacterized Xanthomonadin-B. Salicylic acid had no effect on xanthomonadin biosynthesis. Interference with 3-HBA and 4-HBA biosynthesis also affected X. campestris pv. campestris virulence factor production and reduced virulence in cabbage and Chinese radish. These findings add to our understanding of xanthomonadin biosynthetic mechanisms and further help to elucidate the biological roles of xanthomonadins in X. campestris pv. campestris adaptation and virulence in host plants.