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Acinetobacter baumannii encodes all enzymes required in the tricarboxylic acid (TCA) cycle and glyoxylate bypass except for isocitrate dehydrogenase kinase/phosphatase (IDHKP), which can phosphorylate isocitrate dehydrogenase (IDH) at a substrate-binding Ser site and control the carbon flux in enterobacteria, such as Escherichia coli. The potential kinase was not successfully pulled down from A. baumannii cell lyase; therefore, whether the IDH 1 from A. baumannii (AbIDH1) can be phosphorylated to regulate intracellular carbon flux has not been clarified. Herein, the AbIDH1 gene was cloned, the encoded protein was expressed and purified to homogeneity, and phosphorylation and enzyme kinetics were evaluated in vitro. Gel filtration and SDS-PAGE analyses showed that AbIDH1 is an 83.5 kDa homodimer in solution. The kinetics showed that AbIDH1 is a fully active NADP-dependent enzyme. The Michaelis constant Km is 46.6 (Mn2+) and 18.1 μM (Mg2+) for NADP+ and 50.5 (Mn2+) and 65.4 μM (Mg2+) for the substrate isocitrate. Phosphorylation experiments in vitro indicated that AbIDH1 is a substrate for E. coli IDHKP. The activity of AbIDH1 treated with E. coli IDHKP immediately decreased by 80% within 9 min. Mass spectrometry indicated that the conserved Ser113 of AbIDH1 is phosphorylated. Continuous phosphorylation-mimicking mutants (Ser113Glu and Ser113Asp) lack almost all enzymatic activity. Side-chain mutations at Ser113 (Ser113Thr, Ser113Ala, Ser113Gly and Ser113Tyr) remarkably reduce the enzymatic activity. Understanding the potential of AbIDH1 phosphorylation enables further investigations of the AbIDH1 physiological functions in A. baumannii.

To reveal the potential mechanism and key determinants that contributed to the improved pectinase activity in Aspergillus niger mutant EIMU2, which was previously obtained by UV-mutagenesis from the wild-type A. niger EIM-6. Proteomic analysis for Aspergillus niger EIMU2 by two-dimensional electrophoresis demonstrated that mutant EIMU2 harbored a multiple enzyme system for the degradation of pectin, mainly constituting by main-chain-cleaving enzymes polygalacturonase, pectate lyase, pectinesterase, and some accessory enzymes rhamnogalacturonan lyase and arabinofuranosidase. Further quantitatively differential proteomic analysis revealed that the quantities of four proteins, pectinesterase, rhamnogalacturonan lyase A, DNA-directed RNA polymerase A, and a hypothetical protein in strain EIMU2 were much higher than those in EIM-6. PCR amplification, sequencing and alignment analysis of genes for the two main members of pectin-degrading enzymes, pectate lyase and polygalacturonase showed that their sequences were completely consistent in A. niger EIM-6 and mutant EIMU2. The result demonstrated that the improved pectinase activity by UV-mutagenesis in A. niger EIMU2 was probably contributed to the up-regulated expression of rhamnogalacturonan lyase, or pectinesterase, which resulted in the optimization of synergy amongst different components of pectin-degrading enzymes.

Nanozymes and natural product-derived herbzymes have been identified in different types of enzymes simulating the natural protein-based enzyme function. How to explore and predict enzyme types of novel nanozymes when synthesized remains elusive. An informed analysis might be useful for the prediction. Here, we applied a protein-evolution analysis method to predict novel types of enzymes with experimental validation. First, reported nanozymes were analyzed by chemical classification and nano-evolution. We found that nanozymes are predominantly classified as protein-based EC1 oxidoreductase. In comparison, we analyzed the evolution of protein-based natural enzymes by a phylogenetic tree and the most conserved enzymes were found to be peroxidase and lyase. Therefore, the natural products of Rhizoma polygonati and Goji herbs were analyzed to explore and test the potent new types of natural nanozymes/herbzymes using the simplicity simulation of natural protein enzyme evolution as they contain these conserved enzyme types. The experimental validation showed that the natural products from the total extract of nanoscale traditional Chinese medicine Huangjing (RP, Rhizoma polygonati) from Mount-Tai (Taishan) exhibit fructose-bisphosphate aldolase of lyase while nanoscale Goji (Lycium chinense) extract exhibits peroxidase activities. Thus, the bioinformatics analysis would provide an additional tool for the virtual discovery of natural product nanozymes.

The present study investigated the induction of the glycolysis product methylglyoxal by trimethylamine (TMA) lyase synthesis in the intestinal microbiota and investigated the intervention mechanism of the effects of dietary fiber on methylglyoxal formation. Intestinal digesta samples, collected from the ceca of mice fed with choline-rich and fiber-supplemented diets, were incubated in an anaerobic environment at 37 °C and pH 7.0 with choline, glycine, and methylglyoxal as inductive factors. The differences between the gut microbiota and its metagenomic and metabonomics profiles were determined using 16S rRNA gene sequencing analysis. The results elucidated that the different dietary interventions could induce differences in the composition of the microbiota, gene expression profiles associated with glycine metabolism, and glycolysis. As compared to the gut microbiota of choline-diet fed mice, fiber supplementation effectively altered the composition of the microbiota and inhibited the genes involved in choline metabolism, glycine and methylglyoxal accumulation, and TMA lyase expression, and improved the methylglyoxal utilization by regulating the pathway related to pyruvate production. However, the intervention of exogenous methylglyoxal significantly decreased these effects. These findings successfully revealed the correlations between the TMA lyase expression and glycine level, as well as the inhibitory effects of dietary fiber on the glycine level, thereby highlighting the role of common glycolytic metabolites as a potential target for TMA production.

The effect of wounding intensity on vitamins and antioxidant activities from potato were evaluated. Potatoes were submitted to five levels of wounding intensity (A/W) including the whole peeled potato (CK), slices, pies, shreds and chopped samples. The results showed that vitamins in potato products had different responses to the same wounding intensity. Wounding intensity significantly induced VB₁, VB₂, nicotinic acid and nicotinamide accumulation in potato products, all of which were higher than that of control at each wounding intensity. Slice cutting increased the content of VB₂, VB₅, VB₇ and VB₉ compared with the control. Shreds cutting increased the content of nicotinic acid, nicotinamide and VC compared with the control. Chopped samples significantly increased the content of VB₆, chlorogenic acid and coumaric acid. The wounding intensities also induced the accumulation of L‐phenylalanin ammonia lyase (PAL) activity. Superoxide dismutase (SOD) and peroxidase (POD) activities of potato products decreased after treated by cutting process.

Human cytochrome P450 CYP17A1 first catalyzes hydroxylation at the C17 position of either pregnenolone (PREG) or progesterone (PROG), and a subsequent C(17)–C(20) bond scission to produce dehydroepiandrosterone (DHEA) or androstenedione (AD). In the T306A mutant, replacement of the Threonine 306 alcohol functionality, essential for efficient proton delivery in the hydroxylase reaction, has only a small effect on the lyase activity. In this work, resonance Raman spectroscopy is employed to provide crucial structural insight, confirming that this mutant, with its disordered proton shuttle, fails to generate essential hydroxylase pathway intermediates, accounting for the loss in hydroxylase efficiency. Significantly, a corresponding spectroscopic study with the susceptible lyase substrate, 17-OH PREG, not only reveals an initially trapped peroxo-iron intermediate experiencing an H-bond interaction of the 17-OH group with the proximal oxygen of the Fe-O(p)-O(t) fragment, facilitating peroxo- attack on the C(20) carbon, but also unequivocally shows the presence of the subsequent hemiketal intermediate of the lyase reaction.

The effect of light and cadmium ions (Cd) on the morphophysiological characteristics and accumulation of low molecular weight phenolic antioxidants in the tea plant Camellia sinensis L. callus culture under their separate and combined action is studied. It was established that the light exposure and the cadmium intake separately led to a change in the morphophysiological characteristics of the culture, a decrease in its growth, and activation of the antioxidant system. It was noted that it was manifested in an increase in the total content of phenolic compounds, flavans, and proanthocyanidins (to a lesser extent) and did not depend on the activity of the phenolic metabolism key enzyme, L-phenylalanine ammoniac lyase. It was found that the cell response was more pronounced under the action of Cd than under the action of light. In some cases, these indicators correlated with the level of lipid peroxidation in callus cultures. It is shown that all changes were more pronounced under the combined action of light and Cd.

Alginate lyases play important roles in alginate degradation in the ocean. Although a large number of alginate lyases have been characterized, little is yet known about those in extremely cold polar environments, which may have unique mechanisms for environmental adaptation and for alginate degradation. Here, we report the characterization of a novel PL7 alginate lyase AlyC3 from Psychromonas sp. C-3 isolated from the Arctic brown alga Laminaria, including its phylogenetic classification, catalytic properties, and structure. We propose the establishment of a new PM-specific subfamily of PL7 (subfamily 6) represented by AlyC3 based on phylogenetic analysis and enzymatic properties. Structural and biochemical analyses showed that AlyC3 is a dimer, representing the first dimeric endo-alginate lyase structure. AlyC3 is activated by NaCl and adopts a novel salt-activated mechanism; that is, salinity adjusts the enzymatic activity by affecting its aggregation states. We further solved the structure of an inactive mutant H127A/Y244A in complex with a dimannuronate molecule and proposed the catalytic process of AlyC3 based on structural and biochemical analyses. We show that Arg(82) and Tyr(190) at the two ends of the catalytic canyon help the positioning of the repeated units of the substrate and that His(127), Tyr(244), Arg(78), and Gln(125) mediate the catalytic reaction. Our study uncovers, for the first time, the amino acid residues for alginate positioning in an alginate lyase and demonstrates that such residues involved in alginate positioning are conserved in other alginate lyases. This study provides a better understanding of the mechanisms of alginate degradation by alginate lyases.

Ethylenediamine-N,N'-disuccinic acid (EDDS) is a promising chelating agent for the remediation of heavy metal-contaminated soil. In general, EDDS is produced through a chemical method. In this study, we report an efficient biotechnological approach for EDDS production using an immobilized enzyme. We expressed the EDDS lyase in E. coli and obtained 19.8 g/L of EDDS through a reaction catalyzed by crude enzymes, containing EDDS lyase and fumarase. After performing metal affinity chromatography-mediated purification, we thoroughly eliminated the fumarase activity, which could result in the unnecessary formation of malate. Then, the purified EDDS lyase was immobilized on a glutaraldehyde-activated amino carrier, and the immobilized enzyme was used in 11 batches (864.5 h). After optimization, 209.3 g/L EDDS was obtained in a 100 mL reaction system, resulting in 20.2 g of EDDS product with a purity of 99.8 % after isolation. The yields of reaction and isolation were 94.0 % and 91.8 %, respectively. In conclusion, this study describes a promising bioproduction process for industrial-level EDDS production.

Glyphosate is an important organophosphonate herbicide used to eliminate grasses and herbaceous plants in many vegetation management situations. Its extensive use is causing environmental pollution, and consequently, there is a need to remove it from the environment using an eco-friendly and cost-effective method. As a step to address this problem, a novel bacterial strain Comamonas odontotermitis P2, capable to utilize glyphosate as a carbon (C) and/or phosphorus (P) source, was isolated from a glyphostate-contaminated field soil in Australia and characterized. Response surface methodology (RSM) employing a 23 full factorial central composite design was used to optimize glyphosate degradation by C. odontotermitis P2 under various culture conditions. The strain C. odontotermitis P2 was proficient in degrading 1.5 g L−1 glyphosate completely within 104 h. The optimal conditions for the degradation of glyphosate were found to be pH 7.4, 29.9 °C, and an inoculum density of 0.54 g L−1 resulting in a maximum degradation of 90%. Sequencing of glyphosate oxidoreductase (GOX) and C-P lyase (phnJ) genes from C. odontotermitis P2 revealed 99% and 93% identities to already reported bacterial GOX and phnJ genes, respectively. The presence of these two genes in C. odontotermitis indicates its potential to degrade glyphosate through GOX and C-P lyase metabolic pathways. This study demonstrates the potential of C. odontotermitis P2 for efficient degradation of glyphosate, which can be exploited for remediation of glyphosate.

This is the peer reviewed version of the following article: Discovery of a Histidine‐Based Scaffold as an Inhibitor of Gut Microbial Choline Trimethylamine‐Lyase, which has been published in final form at https://doi.org/10.1002/cmdc.202000571. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. Anaerobic choline metabolism by human gut microbiota to produce trimethylamine (TMA) has recently evolved as a potential therapeutic target because of its association with chronic kidney disease and increased cardiovascular risks. Limited examples of choline analogues have been reported as inhibitors of bacterial enzyme choline TMA‐lyase (CutC), a key enzyme regulating choline anaerobic metabolism. We used a new workflow to discover CutC inhibitors based on focused screening of a diversified library of small molecules for intestinal metabolic stability followed by in vitro CutC inhibitory assay. This workflow identified a histidine‐based scaffold as a CutC inhibitor with an IC50 value of 1.9±0.2 μM. Remarkably, the identified CutC inhibitor was able to reduce the production of TMA in whole‐cell assays using various bacterial strains as well as in complex gut microbiota environment. The improved efficiency of the new scaffold identified in this study in comparison to previously reported CutC inhibitors would enable optimization of potential leads for in vivo screening and clinical translation. Finally, docking studies and molecular‐dynamic simulations were used to predict putative interactions created between inhibitor and CutC.

RecJ reportedly participates in the base excision repair (BER) pathway, but structural and functional data are scarce. Herein, the Deinococcus radiodurans RecJ (drRecJ) deletion strain exhibited extreme sensitivity to hydrogen peroxide and methyl-methanesulphonate, as well as a high spontaneous mutation rate and an accumulation of unrepaired abasic sites in vivo, indicating the involvement of drRecJ in the BER pathway. The binding affinity and nuclease activity preference of drRecJ toward DNA substrates containing a 5′-P-dSpacer group, a 5′-deoxyribose-phosphate (dRP) mimic, were established. A 1.9 Å structure of drRecJ in complex with 5′-P-dSpacer-modified single-stranded DNA (ssDNA) revealed a 5′-monophosphate binding pocket and occupancy of 5′-dRP in the drRecJ nuclease core. The mechanism for RecJ 5′-dRP catalysis was explored using structural and biochemical data, and the results implied that drRecJ is not a canonical 5′-dRP lyase. Furthermore, in vitro reconstitution assays indicated that drRecJ tends to participate in the long-patch BER pathway rather than the short-patch BER pathway.

DNA polymerase (pol) β catalyzes two reactions at DNA gaps generated during base excision repair, gap-filling DNA synthesis and lyase-dependent 5´-end deoxyribose phosphate removal. The lyase domain of pol β has been proposed to function in DNA gap recognition and to facilitate DNA scanning during substrate search. However, the mechanisms and molecular interactions used by pol β for substrate search and recognition are not clear. To provide insight into this process, a comparison was made of the DNA binding affinities of WT pol β, pol λ, and pol μ, and several variants of pol β, for 1-nt-gap-containing and undamaged DNA. Surprisingly, this analysis revealed that mutation of three lysine residues in the lyase active site of pol β, 35, 68, and 72, to alanine (pol β KΔ3A) increased the binding affinity for nonspecific DNA ∼11-fold compared with that of the WT. WT pol μ, lacking homologous lysines, displayed nonspecific DNA binding behavior similar to that of pol β KΔ3A, in line with previous data demonstrating both enzymes were deficient in processive searching. In fluorescent microscopy experiments using mouse fibroblasts deficient in PARP-1, the ability of pol β KΔ3A to localize to sites of laser-induced DNA damage was strongly decreased compared with that of WT pol β. These data suggest that the three lysines in the lyase active site destabilize pol β when bound to DNA nonspecifically, promoting DNA scanning and providing binding specificity for gapped DNA.

Benzenoids are compounds associated with floral and fruity flavours in flowers, fruits and leaves and present a role in hormonal signalling in plants. These molecules are produced by the phenyl ammonia lyase pathway. However, some yeasts can also synthesize them from aromatic amino acids using an alternative pathway that remains unknown. Hanseniaspora vineae can produce benzenoids at levels up to two orders of magnitude higher than Saccharomyces species, so it is a model microorganism for studying benzenoid biosynthesis pathways in yeast. According to their genomes, several enzymes have been proposed to be involved in a mandelate pathway similar to that described for some prokaryotic cells. Among them, the ARO10 gene product could present benzoylformate decarboxylase activity. This enzyme catalyses the decarboxylation of benzoylformate into benzaldehyde at the end of the mandelate pathway in benzyl alcohol formation. Two homologous genes of ARO10 were found in the two sequenced H. vineae strains. In this study, nine other H. vineae strains were analysed to detect the presence and per cent homology of ARO10 sequences by PCR using specific primers designed for this species. Also, the copy number of the genes was estimated by quantitative PCR. To verify the relation of ARO10 with the production of benzyl alcohol during fermentation, a deletion mutant in the ARO10 gene of Saccharomyces cerevisiae was used. The two HvARO10 paralogues were analysed and compared with other α-ketoacid decarboxylases at the sequence and structural level.

Rational metabolic-flow switching, which we proposed recently, is an effective strategy to produce an exogenous high-value natural product using transformed plant cells; the proof of this concept was demonstrated using bamboo (Phyllostachys nigra; Pn) cells as a model system. Pn cells were transformed to express 4-hydroxycinnamoyl-CoA hydratase/lyase of Pseudomonas putida KT2440 (PpHCHL), which catalyzes the formation of 4-hydroxybenzaldehyde and vanillin from p-coumaroyl-CoA and feruloyl-CoA, respectively. The PpHCHL-transformed cells accumulated glucose conjugates of 4-hydroxybenzoic acid and vanillic acid, indicating that the PpHCHL products (aldehydes) were further metabolized by inherent enzymes in the Pn cells. The production titers of 4-hydroxybenzoic acid glucose ester, vanillic acid glucose ester, and 4-hydroxybenzoic acid glucoside reached 1.7, 0.17, and 0.14 g/L at the maximum, respectively. These results proved the versatility of Pn cells for producing vanillin-related compounds based on rational metabolic-flow switching.

BackgroundDocosahexaenoic acid (DHA) is an important omega-3 long-chain polyunsaturated fatty acid that has a variety of physiological functions for infant development and human health. Although metabolic engineering was previously demonstrated to be a highly efficient way to rapidly increase lipid production, metabolic engineering has seldom been previously used to increase DHA accumulation inSchizochytriumspp.ResultsHere, a sensitive β-galactosidase reporter system was established to screen for strong promoters inSchizochytriumsp. Four constitutive promoters (EF-1αp,TEF-1p,ccg1p, andubiquitinp) and one methanol-inducedAOX1promoter were characterized by the reporter system with the promoter activityccg1p> TEF-1p > AOX1p(induced) > EF-1αp > ubiquitinp. With the strong constitutive promoterccg1p,SchizochytriumATP-citrate lyase (ACL) and acetyl-CoA carboxylase (ACC) were overexpressed inSchizochytriumsp. ATCC 20888. The cells were cultivated at 28 °C and 250 rpm for 120 h with glucose as the carbon source. Shake-flask fermentation results showed that the overexpression strains exhibited growth curves and biomass similar to those of the wild-type strain. The lipid contents of the wild-type strain and of the OACL, OACC, and OACL-ACC strains were 53.8, 68.8, 69.8, and 73.0%, respectively, and the lipid yields of the overexpression strains were increased by 21.9, 30.5, and 38.3%, respectively. DHA yields of the wild-type strain and of the corresponding overexpression strains were 4.3, 5.3, 6.1, and 6.4 g/L, i.e., DHA yields of the overexpression strains were increased by 23.3, 41.9, and 48.8%, respectively.ConclusionsAcetyl-CoA and malonyl-CoA are precursors for fatty acid synthesis. ACL catalyzes the conversion of citrate in the cytoplasm into acetyl-CoA, and ACC catalyzes the synthesis of malonyl-CoA from acetyl-CoA. The results demonstrate that overexpression of ACL and ACC enhances lipid accumulation and DHA production inSchizochytriumsp.

The short-term effects of salicylic and p-hydroxybenzoic acids on the tea plant Camellia sinensis (L.) Kuntze for in vitro cultures were studied. It was found that the acids did not change the morphophysiological parameters of the callus cultures. Meanwhile, the content of malondialdehyde and phenolic compounds, including their monomeric (phenylpropanoids and flavans) and oligomeric (proanthocyanidins) forms, decreased with an increase in the amount of lignin (polymer of phenolic nature) in the treated calli. The activity of L-phenylalanine ammonia lyase (the chief enzyme of phenilic metabolism) did not change upon exposure to p-hydroxybenzoic but decreased after contact with salicylic acid.

This work was supported by the BBSRC Grant BB/N006615/1. Body odour is a characteristic trait of Homo sapiens, however its role in human behaviour and evolution is poorly understood. Remarkably, body odour is linked to the presence of a few species of commensal microbes. Herein we discover a bacterial enzyme, limited to odour-forming staphylococci that are able to cleave odourless precursors of thioalcohols, the most pungent components of body odour. We demonstrated using phylogenetics, biochemistry and structural biology that this cysteine-thiol lyase (C-T lyase) is a PLP-dependent enzyme that moved horizontally into a unique monophyletic group of odour-forming staphylococci about 60 million years ago, and has subsequently tailored its enzymatic function to human-derived thioalcohol precursors. Significantly, transfer of this enzyme alone to non-odour producing staphylococci confers odour production, demonstrating that this C-T lyase is both necessary and sufficient for thioalcohol formation. The structure of the C-T lyase compared to that of other related enzymes reveals how the adaptation to thioalcohol precursors has evolved through changes in the binding site to create a constrained hydrophobic pocket that is selective for branched aliphatic thioalcohol ligands. The ancestral acquisition of this enzyme, and the subsequent evolution of the specificity for thioalcohol precursors implies that body odour production in humans is an ancient process. Publisher PDF

Enzymes exercise impeccable control over chemoselectivity, site selectivity and stereoselectivity in reactions they mediate, such that we have witnessed a surge in the development of new biocatalytic methods. Although carbon–carbon (C–C) bonds are the central framework of organic molecules, biocatalytic methods for their formation have largely been limited to a select few lyase enzymes. Thus, despite several decades of research, there are not many biocatalytic C–C-bond-forming transformations at our disposal. This Review describes the suite of enzymes available for highly selective, biocatalytic C–C bond formation. We discuss each class of enzyme in terms of native activity, alteration of this activity through protein or substrate engineering, and its utility in abiotic synthesis. Discovery and application of enzymes that catalyse new reactions is essential for broad implementation of biocatalysts in organic synthesis. This Review describes recent developments in biocatalytic carbon–carbon bond formation.

The gut microbe-derived metabolite trimethylamine-N-oxide (TMAO) has recently been linked to cardiovascular disease (CVD) pathogenesis, prompting the development of therapeutic strategies to reduce TMAO. Previous work has shown that experimental alteration of circulating TMAO levels via dietary alterations or inhibition of the host TMAO producing enzyme flavin containing monooxygenase 3 (FMO3) is associated with reorganization of host cholesterol and bile acid metabolism in mice. In this work, we set out to understand whether recently developed nonlethal gut microbe-targeting small molecule choline trimethylamine (TMA) lyase inhibitors also alter host cholesterol and bile acid metabolism. Treatment of mice with the mechanism-based choline TMA lyase inhibitor, iodomethylcholine (IMC), increased fecal neutral sterol loss in the form of coprostanol, a bacteria metabolite of cholesterol. In parallel, IMC treatment resulted in marked reductions in the intestinal sterol transporter Niemann-pick C1-like 1 (NPC1L1) and reorganization of the gut microbial community, primarily reversing choline supplemented diet-induced changes. IMC also prevented diet-driven hepatic cholesterol accumulation, causing both upregulation of the host hepatic bile acid synthetic enzyme CYP7A1 and altering the expression of hepatic genes critical for bile acid feedback regulation. These studies suggest that the gut microbiota-driven TMAO pathway is closely linked to both microbe and host sterol and bile acid metabolism. Collectively, as gut microbe-targeting choline TMA lyase inhibitors move through the drug discovery pipeline from preclinical models to human studies, it will be important to understand how these drugs impact both microbe and host cholesterol and bile acid metabolism. NEW & NOTEWORTHY The gut microbe-dependent metabolite trimethylamine-N-oxide (TMAO) has been strongly associated with cardiovascular mortality, prompting drug discovery efforts to identify points of therapeutic intervention within the microbe host TMAO pathway. Recently, mechanism-based small molecule inhibitors of the major bacterial trimethylamine (TMA) lyase enzymes have been developed, and these drugs show efficacy as anti-atherothrombotic agents. The novel findings of this study are that small molecule TMA lyase inhibition results in beneficial reorganization of host cholesterol and bile acid metabolism. This study confirms previous observations that the gut microbial TMAO pathway is intimately linked to host cholesterol and bile acid metabolism and provides further rationale for the development of small molecule choline TMA lyase inhibitors for the treatment of cardiometabolic disorders.

Ovothiols are histidine-derived thiols that are receiving a great interest for their biological activities in human model systems. Thanks to the position of the thiol group on the imidazole ring of histidine, these compounds exhibit unusual antioxidant properties. They have been revealing a very promising pharmacological potential due to their anti-proliferative and anti-inflammatory properties, as well as anti-fibrotic activities not always related to their antioxidant power. Ovothiols occur in three differentially methylated forms (A, B and C), isolated from ovary, eggs and biological fluids of many marine invertebrates, mollusks, microalgae, and pathogenic protozoa. These molecules are synthesized by two enzymes: the sulfoxide synthase OvoA and the sulfoxide lyase OvoB. OvoA catalyzes the insertion of the sulfur atom of cysteine on the imidazole ring of histidine, leading to the formation of a sulfoxide intermediate. This is then cleaved by OvoB, giving 5-thiohistidine, finally methylated on the imidazole ring thanks to the methyltransferase domain of OvoA. Recent studies have shown that OvoA homologs are encoded in a wide variety of genomes suggesting that ovothiol biosynthesis is much more widespread in nature than initially thought. Here we have investigated the OvoA occurrence in diatoms, one of the most abundant group of microalgae, dominating marine and freshwater environments. They are considered a very good model system for both biology/photophysiology studies and for biotechnological applications. We have performed comparative sequence and phylogenetic analyses of OvoA from diatoms, highlighting a high degree of conservation of the canonical domain architecture in the analyzed species, as well as a clear clustering of OvoA in the two different morphological groups, i.e. centric and pennate diatoms. The in silico analyses have also revealed that OvoA gene expression is modulated by growth conditions. More importantly, we have characterized the thiol fraction from cultures of the coastal centric diatom Skeletonema marinoi, providing the first evidence of ovothiol B biosynthesis in diatoms.

Several soybean (Glycine max) germplasms, such as Nishiyamahitashi 98-5 (NH), have an intense seaweed-like flavor after cooking because of their high seed S-methylmethionine (SMM) content. In this study, we compared the amounts of amino acids in the phloem sap, leaves, pods, and seeds between NH and the common soybean cultivar Fukuyutaka. This revealed a comparably higher SMM content alongside a higher free Met content in NH seeds, suggesting that the SMM-hyperaccumulation phenotype of NH soybean was related to Met metabolism in seeds. To investigate the molecular mechanism behind SMM hyperaccumulation, we examined the phenotype-associated gene locus in NH plants. Analyses of the quantitative trait loci in segregated offspring of the cross between NH and the common soybean cultivar Williams 82 indicated that one locus on chromosome 10 explains 71.4% of SMM hyperaccumulation. Subsequent fine-mapping revealed that a transposon insertion into the intron of a gene, Glyma.10g172700, is associated with the SMM-hyperaccumulation phenotype. The Glyma.10g172700-encoded recombinant protein showed Met-γ-lyase (MGL) activity in vitro, and the transposon-insertion mutation in NH efficiently suppressed Glyma.10g172700 expression in developing seeds. Exogenous administration of Met to sections of developing soybean seeds resulted in transient increases in Met levels, followed by continuous increases in SMM concentrations, which was likely caused by Met methyltransferase activity in the seeds. Accordingly, we propose that the SMM-hyperaccumulation phenotype is caused by suppressed MGL expression in developing soybean seeds, resulting in transient accumulation of Met, which is converted into SMM to avoid the harmful effects caused by excess free Met.

The first patient suffering from severe acute respiratory syndrome (SARS) was identified in December 2019 in Wuhan, China. Physicians and scientists consequently diagnosed and identified this case of SARS as COVID-19, which was caused by infection with SARS-CoV-2, a new coronavirus. To date, it has spread as a global pandemic, with more than 2.5 million confirmed patients and 175 thousand deaths. Unfortunately, we have yet to find a specific effective therapy; although, some maintenance therapies are known to improve symptoms, partially referencing the experiences from anti-SARS-CoV and the Middle East respiratory syndrome. In addition, many clinical trials are completed or ongoing. Accordingly, a new strategy for development of therapeutic drugs is urgently needed. Here, we propose to prepare a kind of carbon nanotube with functions to exert acidification for cytoplasmic and local cellular temperature-rising through photothermal conversion, according to the physical and chemical nature of carbon nanotubes having been well applied to facilitate such a response. Dexterously, we will put the above effects into practice to inhibit SARS-CoV-2 replication with respect to the biological nature of coronavirus.

Lung disease caused by Pseudomonas aeruginosa is the leading reason for death in cystic fibrosis patients. Therapeutic efficacy of the pharmacological pairs, the naked/encapsulated mutant form of C...

Human O-phosphoethanolamine phospho-lyase (hEtnppl; EC 4.2.3.2) is a pyridoxal 5′-phosphate-dependent enzyme that catalyzes the degradation of O-phosphoethanolamine (PEA) into acetaldehyde, phosphate and ammonia. Physiologically, the enzyme is involved in phospholipid metabolism, as PEA is the precursor of phosphatidylethanolamine in the CDP-ethanolamine (Kennedy) pathway. Here, the crystal structure of hEtnppl in complex with pyridoxamine 5′-phosphate was determined at 2.05 Å resolution by molecular replacement using the structure of A1RDF1 from Arthrobacter aurescens TC1 (PDB entry 5g4i) as the search model. Structural analysis reveals that the two proteins share the same general fold and a similar arrangement of active-site residues. These results provide novel and useful information for the complete characterization of the human enzyme.

p-Coumaric acid (p-CA) is a bioactive natural product and an important industrial material for pharmaceuticals and nutraceuticals. It can be synthesized from deamination of l-tyrosine by tyrosine ammonia lyase (TAL). In this work, we discovered two aromatic amino acid lyase genes, Sas-tal and Sts-tal, from Saccharothrix sp. NRRL B-16348 and Streptomyces sp. NRRL F-4489, respectively, and expressed them in Escherichia coli BL21(DE3). The two enzymes were functionally characterized as TAL. The optimum reaction temperature for Sas-TAL and Sts-TAL is 55 °C and 50 °C, respectively; while, the optimum pH for both TALs is 11. Sas-TAL had a kcat/Km value of 6.2 μM−1 min−1, while Sts-TAL had a much higher efficiency with a kcat/Km value of 78.3 μM−1 min−1. Both Sts-TAL and Sas-TAL can also take l-phenylalanine as the substrate to yield trans-cinnamic acid, and Sas-TAL showed much higher phenylalanine ammonia lyase activity than Sts-TAL. Using E. coli/Sts-TAL as a whole-cell biocatalyst, the productivity of p-CA reached 2.88 ± 0.12 g (L h)−1, which represents the highest efficiency for microbial production of p-CA. Therefore, this work not only reports the identification of two new TALs from actinomycetes, but also provides an efficient way to produce the industrially valuable material p-CA.

Arsenic (As) contamination in paddy soil can cause phytotoxicity and elevated As accumulation in rice grains. Arsenic detoxification is closely linked to sulfur assimilation, but the genes involved have not been described in rice. In this study, we characterize the function of OASTL-A1, an O-acetylserine(thiol) lyase, in cysteine biosynthesis and detoxification of As in rice. Tissue expression analysis revealed that OsOASTL-A1 is mainly expressed in roots at the vegetative growth stage and in nodes at the reproductive stage. Furthermore, the expression of OsOASTL-A1 in roots was strongly induced by As exposure. Transgenic rice plants expressing pOsOASTL-A1::GUS (β-glucuronidase) indicated that OsOASTL-A1 was strongly expressed in the outer cortex and the vascular cylinder in the root mature zone. Subcellular localization using OsOASTL-A1:eGFP (enhanced green fluorescent protein) fusion protein showed that OsOASTL-A1 was localized to the cytosol. In vivo and in vitro enzyme activity assays showed that OsOASTL-A1 possessed the O-acetylserine(thiol) lyase activity. Knockout of OsOASTL-A1 led to significantly lower levels of cysteine, glutathione, and phytochelatins in roots and increased sensitivity to arsenate stress. Furthermore, the osoastl-a1 knockout mutants reduced As accumulation in the roots, but increased As accumulation in shoots. We conclude that OsOASTL-A1 is the cytosolic O-acetylserine(thiol) lyase that plays an important role in non-protein thiol biosynthesis in roots for As detoxification.

This work utilizes X-ray crystallography to provide mechanistic insights into how carbonic anhydrase II can act as a nitrite reductase in the presence of copper., Nitric oxide (NO) promotes vasodilation through the activation of guanylate cyclase, resulting in the relaxation of the smooth muscle vasculature and a subsequent decrease in blood pressure. Therefore, its regulation is of interest for the treatment and prevention of heart disease. An example is pulmonary hypertension which is treated by targeting this NO/vasodilation pathway. In bacteria, plants and fungi, nitrite (NO2 −) is utilized as a source of NO through enzymes known as nitrite reductases. These enzymes reduce NO2 − to NO through a catalytic metal ion, often copper. Recently, several studies have shown nitrite reductase activity of mammalian carbonic anhydrase II (CAII), yet the molecular basis for this activity is unknown. Here we report the crystal structure of copper-bound human CAII (Cu–CAII) in complex with NO2 − at 1.2 Å resolution. The structure exhibits Type 1 (T-1) and 2 (T-2) copper centers, analogous to bacterial nitrite reductases, both required for catalysis. The copper-substituted CAII active site is penta-coordinated with a ‘side-on’ bound NO2 −, resembling a T-2 center. At the N terminus, several residues that are normally disordered form a porphyrin ring-like configuration surrounding a second copper, acting as a T-1 center. A structural comparison with both apo- (without metal) and zinc-bound CAII (Zn–CAII) provides a mechanistic picture of how, in the presence of copper, CAII, with minimal conformational changes, can function as a nitrite reductase.

Tricholoma matsutake is an ectomycorrhizal fungus, related with the host of Pinus densiflora. Most of studies on T. matsutake have focused on mycelial growth, genes and genomics, phylogenetics, symbiosis, and immune activity of this strain. T. matsutake is known for its unique fragrance in Eastern Asia. The most major component of its scent is (R)-(-)-1-octen-3-ol and is biosynthesized from the substrate linoleic acid by the sequential reaction of lipoxygenase and peroxide lyase. Here, we report for the first time the biosynthesis of (R)-(-)- 1-octen-3-ol of T. matsutake using the yeast Saccharomyces cerevisiae as a host. In this study, cDNA genes correlated with these reactions were cloned from T. matsutake, and expression studies of theses genes were carried out in the yeast Saccharomyces cerevisiae. The product of these genes expression study was carried out with Western blotting. The biosynthesis of (R)-(-)- 1-octen-3-ol of T. matsutake in recombinant Saccharomyces cerevisiae was subsequently identified with GC-MS chromatography analysis. The biosynthesis of (R)-(-)-1-octen-3-ol with S. cerevisiae represents a significant step forward.

Cysteine serves as the sulfur source for the biosynthesis of Fe–S clusters and thio-cofactors, molecules that are required for core metabolic processes in all organisms. Therefore, cysteine desulfurases, which mobilize sulfur for its incorporation into thio-cofactors by cleaving the Cα—S bond of cysteine, are ubiquitous in nature. SufS, a type 2 cysteine desulfurase that is present in plants and microorganisms, mobilizes sulfur from cysteine to the transpersulfurase SufE to initiate Fe–S biosynthesis. Here, a 1.5 Å resolution X-ray crystal structure of the Escherichia coli SufS homodimer is reported which adopts a state in which the two monomers are rotated relative to their resting state, displacing a β-hairpin from its typical position blocking transpersulfurase access to the SufS active site. A global structure and sequence analysis of SufS family members indicates that the active-site β-hairpin is likely to require adjacent structural elements to function as a β-latch regulating access to the SufS active site.

The synthesis of complex molecules from simple, renewable carbon units is the goal of a sustainable economy. Here we explored the biocatalytic potential of the thiamine‐diphosphate‐dependent (ThDP) oxalyl‐CoA decarboxylase (OXC)/2‐hydroxyacyl‐CoA lyase (HACL) superfamily that naturally catalyzes the shortening of acyl‐CoA thioester substrates through the release of the C1‐unit formyl‐CoA. We show that the OXC/HACL superfamily contains promiscuous members that can be reversed to perform nucleophilic C1‐extensions of various aldehydes to yield the corresponding 2‐hydroxyacyl‐CoA thioesters. We improved the catalytic properties of Methylorubrum extorquens OXC by rational enzyme engineering and combined it with two newly described enzymes—a specific oxalyl‐CoA synthetase and a 2‐hydroxyacyl‐CoA thioesterase. This enzymatic cascade enabled continuous conversion of oxalate and aromatic aldehydes into valuable (S)‐α‐hydroxy acids with enantiomeric excess up to 99 %., The thiamine‐diphosphate‐dependent (ThDP) oxalyl‐CoA decarboxylase (OXC)/2‐hydroxyacyl‐CoA lyase (HACL) superfamily contains promiscuous members that can perform nucleophilic C1‐extensions of aldehydes to yield 2‐hydroxyacyl‐CoA thioesters. An enzymatic cascade consisting of OXC, oxalyl‐CoA synthetase, and a 2‐hydroxyacyl‐CoA thioesterase converted oxalate and aromatic aldehydes into (S)‐α‐hydroxy acids with enantiomeric excess up to 99 %.

Pyridoxal-5′-phosphate (PLP), the active form of vitamin B(6), is an important and versatile coenzyme involved in a variety of enzymatic reactions, accounting for about 4% of all classified activities. However, the detailed catalytic reaction pathways for PLP-dependent enzymes remain to be explored. Methionine-γ-lyase (MGL), a promising alternative anti-tumor agent to conventional chemotherapies whose catalytic mechanism is highly desired for guiding further development of re-engineered enzymes, was used as a representative PLP-dependent enzyme, and the catalytic mechanism for L-Met elimination by MGL was explored at the first-principles quantum mechanical/molecular mechanical (QM/MM) level with umbrella sampling. The QM/MM calculations revealed that the enzymatic reaction pathway consists of 4 stages for a total of 19 reaction steps with five intermediates captured in available crystal structures. Furthermore, the more comprehensive role of PLP was revealed. Besides the commonly known role of “electron sink”, coenzyme PLP can also assist proton transfer and temporarily store the excess proton generated in some intermediate states by using its hydroxyl group and phosphate group. Thus, PLP is participated in most of the 19 steps. This study not only provided a theoretical basis for further development and re-engineering MGL as a potential anti-tumor agent, but also revealed the comprehensive role of PLP which could be used to explore the mechanisms of other PLP-dependent enzymes.

Sphingosine 1-phosphate (S1P) lyase is an intracellular enzyme that catalyzes the irreversible degradation of S1P and has been suggested as a therapeutic target for the treatment of psoriasis vulgaris. Because S1P induces differentiation of keratinocytes, we examined whether modulation of S1P lyase and altered intracellular S1P levels regulate proliferation and differentiation of human neonatal epidermal keratinocyte (HEKn) cells. To identify the physiological functions of S1P lyase in skin, we inhibited S1P lyase in HEKn cells with an S1P lyase-specific inhibitor (SLI) and with S1P lyase 1 (SGPL1)-specific siRNA (siSGPL1). In HEKn cells, pharmacological treatment with the SLI caused G1 arrest by upregulation of p21 and p27 and induced keratin 1, an early differentiation marker. Similarly, genetic suppression by siSGPL1 arrested the cell cycle at the G1 phase and activated differentiation. In addition, enzyme suppression by siSGPL1 upregulated keratin 1 and differentiation markers including involucrin and loricrin. When hyperproliferation of HEKn cells was induced by interleukin (IL)-17 and IL-22, pharmacologic inhibition of S1P lyase by SLI decreased proliferation and activated differentiation of HEKn cells simultaneously. In addition, SLI administration ameliorated imiquimod-induced psoriatic symptoms including erythema, scaling, and epidermal thickness in vivo. We thus demonstrated that S1P lyase inhibition reduces cell proliferation and induces keratinocyte differentiation, and that inhibition may attenuate psoriasiform changes. Collectively, these findings suggest that S1P lyase is a modulating factor for proliferation and differentiation, and support its potential as a therapeutic target for psoriasis in human keratinocytes.

Interactions of Citrobacter freundii methionine γ-lyase (MGL) with sulfoxides of typical substrates were investigated. It was found that sulfoxides are suicide substrates of the enzyme. The products of the β- and γ-elimination reactions of sulfoxides, thiosulfinates, oxidize three cysteine residues of the enzyme. Three-dimensional structures of MGL inactivated by dimethyl thiosulfinate and diethyl thiosulfinate were determined at 1.46 A and 1.59 A resolution. Analysis of the structures identified SH groups oxidized by thiosulfinates and revealed the structural bases of MGL inactivation. The extent of inactivation of MGL in the catalysis of the β-elimination reaction depends on the length of the «tail» at oxidized Cys115. Oxidation of Cys115 results in MGL incapable to catalyze the stage of methyl mercaptan elimination of the physiological reaction.

Fourteen previously undescribed naturally occurring C-23 carboxylated triterpenoids, stewartiacids A–N (1–14), were isolated and characterized from the twigs and leaves of the ornamental and medicinal plant Stewartia sinensis (Chinese Stewartia), a ‘vulnerable’ species endemic to China. The new structures were elucidated on the basis of spectroscopic data, single crystal X-ray diffraction, and electronic circular dichroism (ECD) analyses. Stewartiacids A (1) and B (2) are isoursenol derivatives. Stewartiacid C (3) is a 12-oxo-γ-amyrin analogue. Both isoursenol and γ-amyrin derivatives are quite rare in nature. Stewartiacids D (4) and E (5) are 13,27-cycloursane-type compounds. Stewartiacids K (11) and L (12) are ursane-type triterpene and phenylpropanol adducts built through a 1,4-dioxane ring, which are also seldom reported in the literature. The rest are common C-23 carboxylated ursane-type (6–10) and oleanane-type (13, 14) pentacyclic triterpenoids. Stewartiacids G (7), K (11), and L (12) showed moderate inhibitory effects against ATP-citrate lyase (ACL), with IC50 values of 12.5, 2.8, and 10.6 μM, respectively. Stewartiacid K (11) also exhibited moderate inhibition (IC50: 16.8 μM) of NF-κB.

Organomercury lyase (MerB) overexpressed in Escherichia coli captured and decomposed organomercury compounds, and it has been detected by radioactive analysis with neutron irradiation. Genetically modified E. coli captures a lot of mercury from a cultivation solution with about 80% recovery, when the bacteria are growing during 24 to 72 hours. Since the modified E. coli has no additive gene for mercury metabolism, the bacteria could hold mercury tightly by the MerB enzyme in their cell and do not release them into medium. In the later, 72 hours after, bacteria have less recovery ratio; it may be affected by undecompsed mercury compounds in bacteria growth. The recovery ability of the bacteria would not be changed by addition of the MerB producing reagent (IPTG). A quantitative value of mercury atom is estimated by an emission of γ-ray by reactor neutron from a dried cell or solution on a filter paper, which is available for nondestructive testing of bacteria holding mercury atoms. In this method an efficient recovery system of toxic mercury from a polluted solution has been archived without destruction of samples, so called in-cell analysis.

Fragaria chiloensis (L.) Mill. fruit has exotic organoleptic properties however commercialization is a challenge due to its fast and intensive softening. Texture modifications associated to ripening are related to cell wall metabolism. Main cell wall polysaccharides metabolized in F. chiloensis fruit are pectins, being rhamnogalacturonan I (RG-I) an abundant pectin domain in strawberry. Several enzymes belonging to the fruit molecular machinery have been described to act on different cell wall polysaccharides in F. chiloensis, but none acting on the main chain of RG-I until now. A gene sequence coding for a rhamnogalacturonan endolyase (RG-lyase) (EC 4.2.2.23) was isolated from F. chiloensis. The FchRGL1 sequence belongs to Polysaccharide Lyase family 4 and contains the three functional domains of RG-lyases: RGL4 domain, fibronectin type III and the carbohydrate binding module. In addition, it contains key amino acid residues for activity and Ca2+ coordination. qRT-PCR analyses indicate that FchRGL1 transcripts increase in fruit throughout ripening. RG-lyase activity evidences a remarkable increase as the fruit ripens. The heterologous expression of FchRGL1 in Pichia pastoris provided an active protein that allows its biochemical characterization. RG-lyase activity is optimum at pH 5.0, 25–30 °C and 2 mM Ca2+. A KM of 0.086 mg mL−1 was determined for potato RG-I, and the enzyme undergoes inhibition at high substrate concentration. The enzyme is also able to degrade the mucilage of germinating A. thaliana's seeds. Finally, the properties of FchRGL1 and its expression pattern are congruent with a crucial role in cell wall re-organization during softening of F. chiloensis fruit.

Objective: The objective of this study was to report CYB5A deficiency, to discuss the contribution of steroid metabolomics to diagnosis and interpretation, and to highlight the presence of testicular microlithiasis. Methods: Two siblings with ambiguous genitalia at birth were later found to carry novel CYB5A variants, with resulting isolated 17, 20 lyase deficiency. We compared urine steroid data obtained between birth and adulthood with that from other cases. Results: Neonatal urine steroid profiles show a relative increase of 16-hydroxylated pregnenolone metabolites. Thereafter, there are no distinguishing features until puberty, when sex steroid deficiency drives gonadotrophin production, resulting in marked increases of 17-hydroxyprogesterone metabolites derived from the gonads. This excess may be revealed pre-pubertally by gonadotrophin stimulation testing. Novel findings are first, a considerable capacity for DHEA synthesis in the neonatal period compared to childhood and adulthood, suggesting that DHEAS production is much less dependent on CYB5A at birth; second, no consistent change in “backdoor pathway” intermediates; third, side chain cleavage of cortisol is largely unaffected, supporting the existence of a different lyase not dependent on CYB5A; fourth, increased 17-hydroxyprogesterone metabolites and very low androgen metabolites are diagnostic post-pubertally. Conclusion: This is the fourth disease-causing variant in CYB5A in isolated 17, 20 lyase deficiency and the first associated with testicular microlithiasis. Establishing a biochemical diagnosis pre-pubertally should now be possible using urine steroid profiling, supported by synacthen and gonadotrophin stimulation testing. We recommend liquid chromatography-mass spectrometry/mass spectrometry rather than immunoassay for serum steroid analysis, early methaemoglobin measurement and surveillance should testicular microlithiasis be detected.

Predicting enzymes function is an important and difficult problem, particularly when enzymes may have the multiplex character, i.e., some enzymes simultaneously have two or three function classes. Most of the existing enzyme function predictor can only be used to deal with the mono-functional enzymes. Actually, multi-functional enzymes should not be ignored because they usually possess diverse biological functions worthy of our special notice. By introducing the “improved Hybrid Multi-label Classifier” and “neighbor score”, a new predictor, called MF-EFP, has been developed that can be used to deal with the systems containing both mono-functional and multi-functional enzymes. As demonstration, the jackknife cross-validation was performed with MF-EFP on a benchmark dataset of enzymes classified into the following 7 functional classes: (1) EC 1 Oxidoreductase, (2) EC 2 Transferase, (3) EC 3 Hydrolase, (4) EC 4 Lyase, (5) EC 5 Isomerase, (6) EC 6 Ligase, (7) EC7 Translocases, where none of enzymes included has ≥90% pairwise sequence identity to any other in a same subset. The subset accuracy and average precision thus obtained by MF-EFP was 85.62% and 94.16% respectively. Extensive experiments also show that MF-EFP can outperform the existing predictors that also have the capacity to deal with such a complicated and stringent system. As a user-friendly web-server, MF-EFP is freely accessible to the public at the web-site http://www.jci-bioinfo.cn/MF-EFP.

Bacterial microcompartments (BMCs) are prokaryotic organelles consisting of a protein shell and an encapsulated enzymatic core. BMCs are involved in several biochemical processes, such as choline, glycerol and ethanolamine degradation and carbon fixation. Since non-native enzymes can also be encapsulated in BMCs, an improved understanding of BMC shell assembly and encapsulation processes could be useful for synthetic biology applications. Here we report the isolation and recombinant expression of BMC structural genes from the Klebsiella pneumoniae GRM2 locus, the investigation of mechanisms behind encapsulation of the core enzymes, and the characterization of shell particles by cryo-EM. We conclude that the enzymatic core is encapsulated in a hierarchical manner and that the CutC choline lyase may play a secondary role as an adaptor protein. We also present a cryo-EM structure of a pT = 4 quasi-symmetric icosahedral shell particle at 3.3 Å resolution, and demonstrate variability among the minor shell forms., Bacterial microcompartments (BMCs) consist of a protein shell and an encapsulated enzymatic core. Here, Kalnins et al. study a BMC from Klebsiella pneumoniae, show that the enzymatic core is encapsulated in a hierarchical manner, and solve the cryo-EM structure of a pT = 4 quasi-symmetric icosahedral shell particle.

The genetic context in bacterial genomes and screening for potential substrates can help identify the biochemical functions of bacterial enzymes. The Gram-negative, strictly anaerobic bacteriumVeillonella rattipossesses a gene cluster that appears to be related tol-fucose metabolism and contains a putative dihydrodipicolinate synthase/N-acetylneuraminate lyase protein (FucH). Here, screening of a library of 2-keto-3-deoxysugar acids with this protein and biochemical characterization of neighboring genes revealed that this gene cluster encodes enzymes in a previously unknown “route I” nonphosphorylatingl-fucose pathway. Previous studies of other aldolases in the dihydrodipicolinate synthase/N-acetylneuraminate lyase protein superfamily used only limited numbers of compounds, and the approach reported here enabled elucidation of the substrate specificities and stereochemical selectivities of these aldolases and comparison of them with those of FucH. According to the aldol cleavage reaction, the aldolases were specific for (R)- and (S)-stereospecific groups at the C4 position of 2-keto-3-deoxysugar acid but had no structural specificity or preference of methyl groups at the C5 and C6 positions, respectively. This categorization corresponded to the (Re)- or (Si)-facial selectivity of the pyruvate enamine on the (glycer)aldehyde carbonyl in the aldol-condensation reaction. These properties are commonly determined by whether a serine or threonine residue is positioned at the equivalent position close to the active site(s), and site-directed mutagenesis markedly modified C4-OH preference and selective formation of a diastereomer. I propose that substrate specificity of 2-keto-3-deoxysugar acid aldolases was convergently acquired during evolution and report the discovery of anotherl-2-keto-3-deoxyfuconate aldolase involved in the same nonphosphorylatingl-fucose pathway inCampylobacter jejuni.

Hydrogen sulfide (H2S) has been regarded as the third gasotransmitter and plays an active role in multiple signaling events of plants and animals. Cysteine desulfhydrases (CDes), including both D- and L-cysteine desulfhydrases (D/L-CDes) that degrade L- or D-cysteine into H2S, pyruvate, and ammonium, are considered the key enzymes responsible for endogenous H2S generation in plants. Several D-CDes are homologous to 1-aminocyclopropane-1-carboxylate deaminase (ACCD) and possess both ACCD and D-CDes activities, thus not a real specific D-CDes. However, little attention had been paid to true D-CDes and little information has been known about this protein in plants. In this study, a putative D-CDes transcript was cloned and characterized from Oryza sativa which encodes a protein with 423 amino acids possessing D-CDes activity and named as OsDCD1. Neither activities of ACCD nor O-acetyl-L-serine (thiol) lyase (OASTL) can be detected from OsDCD1 recombinant protein. For D-Cys, the Km of OsDCD1 is 0.13 ± 0.01 mM and the Vm is 111.55 ± 1.91 units mg−1 of protein. The pH-optimum and temperature-optimum of the OsDCD1 are 8.5 and 35°C, respectively. By site-directed mutagenesis, mutation of S357E or S357E/T589L almost fully abolished the D-CDes activity of OsDCD1, while the T389L mutant retained only partial D-CDes activity by 3.7%, indicating these two amino acid residues play critical roles for the maintenance of OsDCD1 activity. Besides, subcellular localization analysis in rice protoplast revealed that the OsDCD1 localizes in the chloroplast but not mitochondria, which is different from DCD1 in Arabidopsis. The qRT-PCR analysis further showed that the abundance of OsDCD1 transcript was widely regulated by different hormones and chemical reagents we used. In general, our results provided evidence that OsDCD1 is a potentially important endogenous H2S producing enzyme in rice, which may play an important role in plant growth regulators and chemical stimuli.

Cysteine biosynthesis is directed by the successive commitments of serine acetyltransferase, and O-acetylserine (thiol) lyase (OASTL) compounds, which subsequently frame the decameric cysteine synthase complex. The isoforms of OASTL are found in three compartments of the cell: the cytosol, plastid, and mitochondria. In this investigation, we first isolated putative chloroplastic OASTL (Ch-OASTL) from Leucaena leucocephala, and the Ch-OASTL was then expressed in BL21-competent Escherichia coli. The putative Ch-OASTL cDNA clone had 1,543 base pairs with 391 amino acids in its open reading frame and a molecular weight of 41.54 kDa. The purified protein product exhibited cysteine synthesis ability, but not mimosine synthesis activity. However, they both make the common α-aminoacrylate intermediate in their first half reaction scheme with the conventional substrate O-acetyl serine (OAS). Hence, we considered putative Ch-OASTL a cysteine-specific enzyme. Kinetic studies demonstrated that the optimum pH for cysteine synthesis was 7.0, and the optimum temperature was 40 °C. In the cysteine synthesis assay, the Km and kcat values were 838 ± 26 µM and 72.83 s−1 for OAS, respectively, and 60 ± 2 µM and 2.43 s−1 for Na2S, respectively. We can infer that putative Ch-OASTL regulatory role is considered a sensor for sulfur constraint conditions, and it acts as a forerunner of various metabolic compound molecules.

In the biosynthesis of the macrolactam antibiotic cremimycin, the 3-aminononanoic acid starter unit is formed via a non-2-enoyl acyl carrier protein thioester intermediate, which is presumed to be constructed by cis-acyltransferase (AT) polyketide synthases (PKSs) CmiP2, CmiP3, and CmiP4. While canonical cis-AT PKS modules are comprised of a single polypeptide, the PKS module formed by CmiP2 and CmiP3 is split within the dehydratase (DH) domain. Here, we report the enzymatic function and the structural features of this split-DH domain. In vitro analysis showed that the split-DH domain catalyzes the dehydration reaction of (R)-3-hydroxynonanoyl N-acetylcysteamine thioester (SNAC) to form (E)-non-2-enoyl-SNAC, suggesting that the split-DH domain is catalytically active in cremimycin biosynthesis. In addition, structural analysis revealed that the CmiP2 and CmiP3 subunits of the split-DH domain form a tightly associated heterodimer through several hydrogen bonding and hydrophobic interactions, which are similar to those of canonical DH domains of other cis-AT PKSs. These results indicate that the split-DH domain has the same function and structure as common cis-AT PKS DH domains.

Thiocyanate (SCN-) is harmful to a wide range of organisms, and its removal is essential for environmental protection. A neutrophilic halophile capable of thiocyanate degradation, Thiohalobacter sp. strain FOKN1, was highly enriched (relative abundance; 98.4%) from activated sludge collected from a bioreactor receiving thiocyanate-rich wastewater. The enrichment culture degraded 3.38 mM thiocyanate within 140 h, with maximum activity at pH 8.8, 37°C, and 0.18 M sodium chloride. Thiocyanate degradation was inhibited by 30 mg L-1 phenol, but not by thiosulfate. Microbial thiocyanate degradation is catalyzed by thiocyanate dehydrogenase, while limited information is currently available on the molecular mechanisms underlying thiocyanate degradation by the thiocyanate dehydrogenase of neutrophilic halophiles. Therefore, (meta)genomic and proteomic analyses of enrichment cultures were performed to elucidate the whole genome sequence and proteome of Thiohalobacter sp. strain FOKN1. The 3.23-Mb circular Thiohalobacter sp. strain FOKN1 genome was elucidated using a PacBio RSII sequencer, and the expression of 914 proteins was identified by tandem mass spectrometry. The Thiohalobacter sp. strain FOKN1 genome had a gene encoding thiocyanate dehydrogenase, which was abundant in the proteome, suggesting that thiocyanate is degraded by thiocyanate dehydrogenase to sulfur and cyanate. The sulfur formed may be oxidized to sulfate by the sequential oxidation reactions of dissimilatory sulfite reductase, adenosine-5'-phosphosulfate reductase, and dissimilatory ATP sulfurylase. Although the Thiohalobacter sp. strain FOKN1 genome carried a gene encoding cyanate lyase, its protein expression was not detectable. The present study advances the understanding of the molecular mechanisms underlying thiocyanate degradation by the thiocyanate dehydrogenase of neutrophilic halophiles.

Recent studies revealed that some intestinal microorganisms anaerobically convert choline to trimethylamine (TMA) by choline TMA-lyase (cutC). TMA is further oxidized to trimethylamine-N-oxide (TMAO), by the liver enzyme flavin-dependent monooxygenase 3 (FMO3). TMA in the serum is correlated with the risk of cardiovascular disease and some other diseases in human. The objective of this study is to study the expression levels of cutC and its activating enzyme (cutD) gene for these microorganisms and their association with TMA production. In this study, we collected 20 TMA producing bacteria strains representing 20 species, and designed primers to evaluate their gene expression levels by reverse transcription quantitative PCR (RT-qPCR). In addition, TMA production was analyzed by UPLC-MS/MS. Results showed that gene expression levels of most individual strains were different when compared with the gene expression level of their glyceraldehyde-3 phosphate dehydrogenase (GAPDH) gene and the TMA production level of gut bacteria may not correlate with their cutC/cutD gene expression levels. Bioinformatic analysis of the CutC protein showed conserved choline binding site residues; cutD showed conserved S-adenosylmethionine (SAM) and two CX2-CX2-CX3 motifs. The present study reports that the TMA production level may not only depend on cutC/cutD gene expression. Other factors may need to be investigated.

Resumen La proteína fenilalanina amonio-liasa o PAL es una enzima clave en la ruta de síntesis de los flavonoides; en fresa se han reportado 6 genes que la codifican, entre ellos el FaPAL2. Los flavonoides son metabolitos secundarios que participan en la protección contra luz UV de las plantas, además, son de gran interés farmacéutico debido a las propiedades antioxidantes, antibacterianas, antiinflamatorias, antimutagénicas y anticancerígenas que poseen. Se ha correlacionado el aumento de flavonoides en fresas irradiadas con luz UV-C con altos niveles de expresión del gen FaPAL. Para poder estudiar y controlar la expresión de genes de interés es indispensable conocer la funcionalidad de los promotores, por lo que la presente investigación se planteó por objetivo identificar y aislar el promotor del gen FaPAL2 mediante la técnica TAIL PCR, para posteriormente evaluar su actividad ante respuesta a la luz UV-C en frutos de Fragaria x ananassa cv. ''Camino Real'' vía Agrobacterium tumefaciens utilizando el gen reportero GUS. Se consiguió aislar y secuenciar el promotor del gen FaPAL2, para después generar un constructo genético y evaluar su expresión genética transitoria en frutos agroinfiltrados de fresa. Se identificó una tinción histológica positiva de los frutos agroinfiltrados, tanto irradiados como no irradiados, lo que indica que el promotor del gen FaPAL2 actúa positivamente en respuesta a luz UV-C, pero no de manera exclusiva. Abstract The phenylalanine ammonium lyase protein or PAL is a key enzyme in the pathway of flavonoid synthesis; in strawberry 6 genes have been reported that encode it, including FaPAL2. Flavonoids are essential secondary metabolites for protection against UV light in plants, furthermore, they are of great pharmaceutical interest due to their antioxidants, antibacterial, anti-inflammatory, antimutagenic and anticancer properties. Increase in flavonoid compounds in strawberries irradiated with UV-C light has been correlated with high levels of FaPAL gene expression. In order to study and control the expression of genes of interest, it is indispensable to know the promoters functionality, so the present investigation aimed to identify and isolate the FaPal2 gene promoter using the TAIL PCR technique, to later assess its activity upon response to UV-C light in Fragaria x ananassa cv. ''Camino Real'' fruits via Agrobacterium tumefaciens using the GUS reporter gene. The promoter of the FaPAL2 gene was successfully isolated and sequenced, and later used to generate a genetic construct and evaluate its transient genetic expression in agro-infiltrated strawberry fruits. A positive histological staining was identified in the agro-infiltrated fruits, both irradiated and non-irradiated, indicating that the FaPAL2 gene promoter acts positively in response to UV-C light, but not in an exclusive manner.