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Phycoerythrin (PE) present in the distal ends of light-harvesting phycobilisome rods in Fremyella diplosiphon (Tolypothrix sp. PCC 7601) contains five phycoerythrobilin (PEB) chromophores attached to six cysteine residues for efficient green light capture for photosynthesis. Chromophore ligation on PE subunits occurs through bilin lyase catalyzed reactions, but the characterization of the roles of all bilin lyases for phycoerythrin is not yet complete. To gain a more complete understanding about the individual functions of CpeZ and CpeY in PE biogenesis in cyanobacteria, we examined PE and phycobilisomes purified from wild type F. diplosiphon, cpeZ and cpeY knockout mutants. We find that the cpeZ and cpeY mutants accumulate less PE than wild type cells. We show that in the cpeZ mutant, chromophorylation of both PE subunits is affected, especially the Cys-80 and Cys-48/Cys-59 sites of CpeB, the beta-subunit of PE. The cpeY mutant showed reduced chromophorylation at Cys-82 of CpeA. We also show that, in vitro, CpeZ stabilizes PE subunits and assists in refolding of CpeB after denaturation. Taken together, we conclude that CpeZ acts as a chaperone-like protein, assisting in the folding/stability of PE subunits, allowing bilin lyases such as CpeY and CpeS to attach PEB to their PE subunit.

Alginate lyases have a wide range of industrial applications, such as oligosaccharide preparation, medical treatment, and bioconversion. Therefore, the discovery and characterization of novel alginate lyases are extremely important. PL-6 alginate lyases are classified into two groups: those with a single domain or two domains. However, only one structure of a two-domain alginate lyase has been determined to date. In this study, we characterized a novel single-domain PL-6 alginate lyase (named AlyF). According to the biochemical analysis, AlyF possesses unique features compared with other PL-6 enzymes, including (1) a Ca2+-independent catalytic mechanism and (2) a PolyG-specific cleavage specificity that predominantly produces trisaccharides. The structures of AlyF and its complexes described here reveal the structural basis for these unique features and substrate binding mechanisms, which were further confirmed using mutagenesis. More importantly, we determined the possible subsites specifying the predominantly trisaccharide products of AlyF, which may facilitate the rational design of AlyF for potential applications in preparing a single alginate oligomer.

Phosphoketolases (PK) are TPP-dependent enzymes which play essential roles in carbohydrate metabolism of numerous bacteria. Depending on the substrate specificity PKs can be subdivided into xylulose 5-phosphate (X5P) specific PKs (XPKs) and PKs which accept both X5P and fructose 6-phosphate (F6P) (XFPKs). Despite their key metabolic importance, so far only the crystal structures of two XFPKs have been reported. There are no reported structures for any XPKs and for any complexes between PK and substrate. One of the major unknowns concerning PKs mechanism of action is related to the structural determinants of PKs substrate specificity for X5P or F6P. We report here the crystal structure of XPK from Lactococcus lactis (XPK-Ll) at 2.1 A resolution. Using small angle X-ray scattering (SAXS) we proved that XPK-Ll is a dimer in solution. Towards better understanding of PKs substrate specificity, we performed flexible docking of TPP-X5P and TPP-F6P on crystal structures of XPK-Ll, two XFPKs and transketolase (TK). Calculated structure-based binding energies consistently support XPK-Ll preference for X5P. Analysis of structural models thus obtained show that substrates adopt moderately different conformation in PKs active sites following distinct networks of polar interactions. Based on the here reported structure of XPK-Ll we propose the most probable amino acid residues involved in the catalytic steps of reaction mechanism. Altogether our results suggest that PKs substrate preference for X5P or F6P is the outcome of a fine balance between specific binding network and dissimilar catalytic residues depending on the enzyme (XPK or XFPK) – substrate (X5P or F6P) couples.

N‐cycloalkyl‐substituted amino acids have wide‐ranging applications in pharma‐ and nutraceutical fields. Here we report the asymmetric synthesis of various N‐cycloalkyl‐substituted L‐aspartic acids using ethylenediamine‐N,N'‐disuccinic acid lyase (EDDS lyase) and a previously engineered variant of methylaspartate ammonia lyase (MAL‐Q73A) as biocatalysts. Particularly, EDDS lyase shows broad non‐natural substrate promiscuity and excellent enantioselectivity, allowing the selective addition of homo‐ and heterocycloalkyl amines (comprising four‐, five‐ and six‐membered rings) to fumarate, giving the corresponding N‐cycloalkyl‐substituted L‐aspartic acids with >99% e.e. This biocatalytic methodology offers an alternative synthetic choice to prepare difficult N‐cycloalkyl‐substituted amino acids. Given its very broad amine scope, EDDS lyase is an exceptionally powerful synthetic tool that nicely complements the rapidly expanding toolbox of biocatalysts for asymmetric synthesis of noncanonical amino acids.

Lanthipeptides, which belong to the superfamily of ribosomally synthesized and posttranslationally modified peptides (RiPPs), are associated with various interesting biological activities. Lanthipeptides can be subdivided into four classes that are defined by the characteristics of the corresponding posttranslational modification enzymes. Class IV lanthipeptide synthetases consist of an N-terminal lyase, a central kinase, and a C-terminal cyclase domain. Here, we present the first in-depth characterization of such a kinase domain from the globisporin maturation enzyme SgbL that originates from Streptomyces globisporus sp. NRRL B-2293. Catalytic residues were identified by alignments with homologues and structural modeling. Their roles were confirmed by employing proteins with Ala substitutions in in vitro modification and fluorescence polarization binding assays. Furthermore, the protein region that is binding the leader peptide was identified by hydrogen-deuterium exchange-mass spectrometry experiments. By fusion of this protein region to the maltose binding protein, a protein was generated that can specifically bind the SgbA leader peptide, albeit with reduced binding affinity compared to that of full length SgbL. Combined, the results of this study provide a firmer grasp of how lanthipeptide biosynthesis is accomplished by class IV synthetases and suggest by homology analysis that biosynthetic mechanisms are similar in class III lanthipeptide processing enzymes.

ATP-citrate lyase (ACL) plays a key role in the formation of acetyl-CoA, a crucial precursor for Monascus pigments biosynthesis. Whether ACL affects the pigments synthesis pathway in Monascus spp. is unknown. Here, we focus on acl1 and acl2 genes, encoding two subunits of ACL that were overexpressed in Monascus ruber CICC41233 to generate Monascus ruber ACL438469-4 and Monascus ruber ACL501969-11, respectively. Both acl overexpression strains significantly increased ACL activity, leading to elevate levels of acetyl-CoA. These strains completely degraded starch from rice powder after 4 days. Total Monascus pigments production was remarkably enhanced by 92.70% in M. ruber ACL438469-4 and 112.58% in M. ruber ACL501969-11 after 6 days. The red pigments in M. ruber ACL438469-4 and in M. ruber ACL501969-11 were more than that in parent strain. Transcriptome sequencing analysis of the acl overexpression strains vs the wild-type showed that differentially expressed genes were mainly involved in starch and glycolysis metabolism; valine, leucine, and isoleucine degradation; glycine, serine, and threonine metabolism; and Monascus pigment biosynthetic pathway. This implied metabolic flux for acetyl-CoA during pigment synthesis and NH3 for amination of red pigments. Thus, ACL modulated starch and amino acid metabolism to regulate Monascus pigments production in M. ruber.

Summary In tropical and subtropical oceanic surface waters phosphate scarcity can limit microbial productivity. However, these environments also have bioavailable forms of phosphorus incorporated into dissolved organic matter (DOM) that microbes with the necessary transport and hydrolysis metabolic pathways can access to supplement their phosphorus requirements. In this study we evaluated how the environment shapes the abundance and taxonomic distribution of the bacterial carbon–phosphorus (C–P) lyase pathway, an enzyme complex evolved to extract phosphate from phosphonates. Phosphonates are organophosphorus compounds characterized by a highly stable C–P bond and are enriched in marine DOM. Similar to other known bacterial adaptions to low phosphate environments, C–P lyase was found to become more prevalent as phosphate concentrations decreased. C–P lyase was particularly enriched in the Mediterranean Sea and North Atlantic Ocean, two regions that feature sustained periods of phosphate depletion. In these regions, C–P lyase was prevalent in several lineages of Alphaproteobacteria (Pelagibacter, SAR116, Roseobacter and Rhodospirillales), Gammaproteobacteria, and Actinobacteria. The global scope of this analysis supports previous studies that infer phosphonate catabolism via C–P lyase is an important adaptive strategy implemented by bacteria to alleviate phosphate limitation and expands the known geographic extent and taxonomic affiliation of this metabolic pathway in the ocean.

Phycobilin lyase plays an important role in the binding of phycobilin chromophores to phycobiliprotein. The bilin lyase cpeT gene was cloned from Gracilariopsis lemaneiformis to study the synthesis of fluorescent phycobiliprotein. Two recombinant plasmids, one containing the genes (ho1 and pebA, pebB) that produce phycoerythrobilin and the other containing the two subunit genes (rpeB, rpeA) of phycoerythrin (R-PE), as well as the lyase gene cpeT, were constructed and transformed into Escherichia coli. The recombinant strain with lyase CpeT and the β-subunit of phycoerythrin (R-PeB) enhanced the optical activity of R-PE, indicating that lyase CpeT catalyzes the synthesis of the R-PeB with optical activity. Another two recombinant plasmids, one containing the genes (ho(314), pcyA(314)) that produce phycocyanobilin and the other containing the two subunit genes (rpcB, rpcA) of phycocyanin (R-PC), as well as the lyase gene cpeT, were constructed and transformed into Escherichia coli. However, there was no enhancement in fluorescence in this recombinant strain, revealing that lyase CpeT cannot catalyze the synthesis of optically active R-PC. Thereafter, Cys-82 and Cys-158 of R-PeB were individually mutated to identify the possible binding sites of apoproteins with phycobilins catalyzed by CpeT. We found that there was a significant decrease in the fluorescence of the mutated Cys-158 recombinant strain, suggesting that Cys-158 is the active site for the binding of the β-subunit with CpeT-catalyzed phycoerythrobilin in G. lemaneiformis. The results of this study lay the foundation for understanding the synthesis of fluorescent phycobiliprotein.

Simultaneous production of alkaline xylanase and all seven types of pectinases by a bacterial isolate, under solid-state fermentation was checked in this study. Under optimized conditions, high concurrent production of xylanase (22,800 ± 578 IU/g substrate) and pectinase (4,832 ± 189 IU/g substrate) was achieved. The different types of pectinases produced were exo-polymethylgalacturonase (782 IU/g), endo-polymethylgalacturonase (6.42 U/g), exo-polygalacturonase (2,250 IU/g), endo-polygalacturonase (11.57 U/g), polymethylgalacturonate lyase (53.99 IU/g), polygalacturonate lyase (59.78 IU/g), and pectin esterase (5.78 IU/g). Wheat bran resulted in the highest titer of both enzymes. The maximum xylanase-pectinase yield was detected after 7 days of incubation with 2 mM MgSO4 and 1.5 g/L K2 HPO4 at wheat bran to moisture ratio 1:1.5 (w/v), media to flask volume ratio 1:25, pH 7.0, temperature 37 °C, and inoculum size 15%. Xylanase was most stable at pH 8.0, retained more than 75% activity up to 24 H, whereas pectinase was most stable at pH 9.0, having full activity even after 24 H. At 45 °C, the xylanase showed 82% residual activity after 6 H of incubation. The pectinase was 97% and 61% stable up to 3 H at 50 and 55 °C, respectively. This is the first report showing the production of xylanase-pectinases by bacterium along with high titer of seven types of pectinases, suitable for industries.

Shewanella oneidensis respires a variety of terminal electron acceptors, including solid phase Fe(III) oxides. S. oneidensis transfers electrons to Fe(III) oxides via direct (outer membrane- or nan...

ATP-citrate lyase (ACLY) is a major source of nucleocytosolic acetyl-CoA, a fundamental building block of carbon metabolism in eukaryotes. ACLY is aberrantly regulated in many cancers, cardiovascular disease, and metabolic disorders. However, the molecular mechanisms determining ACLY activity and function are unclear. To this end, we investigated the role of the uncharacterized ACLY C-terminal citrate synthase homology domain in the mechanism of acetyl-CoA formation. Using recombinant, purified ACLY and a suite of biochemical and biophysical approaches, including analytical ultracentrifugation, dynamic light scattering, and thermal stability assays, we demonstrated that the C terminus maintains ACLY tetramerization, a conserved and essential quaternary structure in vitro and likely also in vivo. Furthermore, we show that the C terminus, only in the context of the full-length enzyme, is necessary for full ACLY binding to CoA. Together, we demonstrate that ACLY forms a homotetramer through the C terminus to facilitate CoA binding and acetyl-CoA production. Our findings highlight a novel and unique role of the C-terminal citrate synthase homology domain in ACLY function and catalysis, adding to the understanding of the molecular basis for acetyl-CoA synthesis by ACLY. This newly discovered means of ACLY regulation has implications for the development of novel ACLY modulators to target acetyl-CoA–dependent cellular processes for potential therapeutic use.

The signaling lipid sphingosine 1-phosphate (S1P) plays key roles in many physiological processes. In the immune system, S1P's best-described function is to draw cells out of tissues into circulation. Here, we will review models of S1P distribution in the thymus, lymph nodes, spleen, and nonlymphoid tissues. These models have been challenging to construct, because of the lack of tools to map lipid gradients. Nonetheless, evidence to date suggests that S1P distribution is exquisitely tightly controlled, and that concentrations of signaling-available S1P cannot be predicted by standard rules of thumb. The fine regulation of S1P gradients may explain how S1P can simultaneously direct multiple cell movements both between tissues and circulation and within tissues. It may also make it feasible to develop drugs that enable spatially specific modulation of S1P signaling.

ATP-citrate lyase (ACLY) is a central metabolic enzyme and catalyses the ATP-dependent conversion of citrate and coenzyme A (CoA) to oxaloacetate and acetyl-CoA1–5. The acetyl-CoA product is crucial for the metabolism of fatty acids6,7, the biosynthesis of cholesterol8, and the acetylation and prenylation of proteins9,10. There has been considerable interest in ACLY as a target for anti-cancer drugs, because many cancer cells depend on its activity for proliferation2,5,11. ACLY is also a target against dyslipidaemia and hepatic steatosis, with a compound currently in phase 3 clinical trials4,5. Many inhibitors of ACLY have been reported, but most of them have weak activity5. Here we report the development of a series of low nanomolar, small-molecule inhibitors of human ACLY. We have also determined the structure of the full-length human ACLY homo-tetramer in complex with one of these inhibitors (NDI-091143) by cryo-electron microscopy, which reveals an unexpected mechanism of inhibition. The compound is located in an allosteric, mostly hydrophobic cavity next to the citrate-binding site, and requires extensive conformational changes in the enzyme that indirectly disrupt citrate binding. The observed binding mode is supported by and explains the structure–activity relationships of these compounds. This allosteric site greatly enhances the ‘druggability’ of ACLY and represents an attractive target for the development of new ACLY inhibitors. The structure of human ATP-citrate lyase, in complex with a newly developed small-molecule inhibitor, shows extensive conformational changes that reveal an allosteric site for the inhibitor to bind and indirectly compete with the citrate substrate.

Malonyl-thioesters are reactive centers of malonyl-CoA and malonyl- S-acyl carrier protein, essential to fatty acid, polyketide and various specialized metabolite biosynthesis. Enzymes that create or use malonyl-thioesters spontaneously hydrolyze or decarboxylate reactants on the crystallographic time frame preventing determination of structure-function relationships. To address this problem, we have synthesized a panel of methylmalonyl-CoA analogs with the carboxylate represented by a sulfonate or nitro and the thioester retained or represented by an ester or amide. Structures of Escherichia coli methylmalonyl-CoA decarboxylase in complex with our analogs affords insight into substrate binding and the catalytic mechanism. Counterintuitively, the negatively charged sulfonate and nitronate functional groups of our analogs bind in an active site hydrophobic pocket. Upon decarboxylation the enolate intermediate is protonated by a histidine preventing CO2-enolate recombination, yielding propionyl-CoA. Activity assays support a histidine catalytic acid and reveal the enzyme displays significant hydrolysis activity. Our structures also provide insight into this hydrolysis activity. Our analogs inhibit decarboxylation/hydrolysis activity with low micromolar Ki values. This study sets precedents for using malonyl-CoA analogs with carboxyate isosteres to study the complicated structure-function relationships of acyl-CoA carboxylases, trans-carboxytransferases, malonyltransferases and β-ketoacylsynthases.

Rhizocticins are phosphono-oligopeptide antibiotics that contain a toxic C-terminal ( Z) -l -2-amino-5-phosphono-3-pentenoic acid (APPA) moiety. APPA is an irreversible inhibitor of threonine synthase (ThrC), a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the conversion of O-phospho-l-homoserine to l-threonine. ThrCs are essential for the viability of bacteria, plants, and fungi and are a target for antibiotic development, as de novo threonine biosynthetic pathway is not found in humans. Given the ability of APPA to interfere in threonine metabolism, it is unclear how the producing strain B. subtilis ATCC 6633 circumvents APPA toxicity. Notably, in addition to the housekeeping APPA-sensitive ThrC ( BsThrC), B. subtilis encodes a second threonine synthase (RhiB) encoded within the rhizocticin biosynthetic gene cluster. Kinetic and spectroscopic analyses show that PLP-dependent RhiB is an authentic threonine synthase, converting O-phospho-l-homoserine to threonine with a catalytic efficiency comparable to BsThrC. To understand the structural basis of inhibition, we determined the crystal structure of APPA bound to the housekeeping BsThrC, revealing a covalent complex between the inhibitor and PLP. Structure-based sequence analyses reveal structural determinants within the RhiB active site that contribute to rendering this ThrC homologue resistant to APPA. Together, this work establishes the self-resistance mechanism utilized by B. subtilis ATCC 6633 against APPA exemplifying one of many ways by which bacteria can overcome phosphonate toxicity.

Sphingosine phosphate lyase (SPL) is the final enzyme in the sphingolipid degradative pathway, catalyzing the irreversible cleavage of long-chain base phosphates (LCBPs) to yield a long-chain aldehyde and ethanolamine phosphate (EP). SPL guards the sole exit point of sphingolipid metabolism. Its inactivation causes product depletion and accumulation of upstream sphingolipid intermediates. The main substrate of the reaction, sphingosine-1-phosphate (S1P), is a bioactive lipid that controls immune-cell trafficking, angiogenesis, cell transformation, and other fundamental processes. The products of the SPL reaction contribute to phospholipid biosynthesis and programmed cell-death activation. The main features of SPL enzyme activity were first described in detail by Stoffel et al. in 1969. The first SPL-encoding gene was cloned from budding yeast in 1997. Reverse and forward genetic strategies led to the rapid identification of other genes in the pathway and their homologs in other species. Genetic manipulation of SPL-encoding genes in model organisms has revealed the contribution of sphingolipid metabolism to development, physiology, and host-pathogen interactions. In 2017, recessive mutations in the human SPL gene SGPL1 were identified as the cause of a novel inborn error of metabolism associated with nephrosis, endocrine defects, immunodeficiency, acanthosis, and neurological problems. We refer to this condition as SPL insufficiency syndrome (SPLIS). Here, we share our perspective on the 50-year history of SPL from discovery to disease, focusing on insights provided by model organisms regarding the pathophysiology of SPLIS and how SPLIS raises the possibility of a hidden role for sphingolipids in other disease conditions.

Cysteine functions not only as an amino acid in proteins but also as a precursor for a large number of essential biomolecules. Cysteine is synthesized via the incorporation of sulfide to O-acetylserine under the catalysis of O-acetylserine(thiol)lyase (OASTL). In dicotyledonous Arabidopsis, nine OASTL genes have been reported. However, in their null mutants, only the mutant of CS26 encoding S-sulfocysteine synthase showed the visible phenotypic changes, displaying significantly small plants and pale-green leaves under long-day condition but not short-day condition. Up to now, no OASTL gene or mutant has been identified in monocotyledon. In this study, we isolated a green-revertible albino mutant gra78 in rice (Oryza sativa). Its albino phenotype at the early seedling stage was sensitive to temperature but independent of photoperiod. Map-based cloning revealed that candidate gene LOC_Os01g59920 of GRA78 encodes a putative S-sulfocysteine synthase showing significant similarity with Arabidopsis CS26. Complementation experiment confirmed that mutation in LOC_Os01g59920 accounted for the mutant phenotype of gra78. GRA78 is constitutively expressed in all tissues and its encoded protein is targeted to the chloroplast. In addition, qRT-PCR suggested that expression levels of four OASTL homolog genes and five photosynthetic genes were remarkably down-regulated.

It was established that the functioning of ATP-citrate lyase in maize (Zea mays L.) leaves is regulated by the light conditions of plants, in particular, irradiation of maize plants with red or blue light caused a sharp increase in enzyme activity compared with the “darkness-” and irradiation with far-red light variants. It was shown that active forms of phytochrome and cryptochrome are involved in increase of ATP-citrate lyase activity by regulating the expression of the csy3 gene. This was probably achieved by interacting with a specific G-site of the csy3 gene promoter. Analysis of the nucleotide sequence of the ATP-citrate lyase gene promoter showed the presence of CpG islands, providing the potential for methylation. The study of the methylation status of individual CG dinucleotides of the csy3 gene promoter revealed a change of this index depending on the conditions of the light regime in plants. A high degree of methylation of CG dinucleotides in the csy3 gene promoter resulted in a decrease in the level of transcripts of this gene, and, as a result, inhibition of ATP-citrate lyase activity in the dark and during irradiation with far-red light.

Methylphosphonic acid is synthesized by marine bacteria and is a prominent component of dissolved organic phosphorus. Consequently, methylphosphonic acid also serves as a source of inorganic phosphate (Pi) for marine bacteria that are starved of this nutrient. Conversion of methylphosphonic acid into Pi is currently only known to occur through the carbon-phosphorus lyase pathway, yielding methane as a byproduct. In this work, we describe an oxidative pathway for the catabolism of methylphosphonic acid in Gimesia maris DSM8797. G. maris can use methylphosphonic acid as Pi sources despite lacking a phn operon encoding a carbon-phosphorus lyase pathway. Instead, the genome contains a locus encoding homologues of the non-heme Fe(II) dependent oxygenases HF130PhnY* and HF130PhnZ, which were previously shown to convert 2-aminoethylphosphonic acid into glycine and Pi. GmPhnY* and GmPhnZ1 were produced in E. coli and purified for characterization in vitro. The substrate specificities of the enzymes were evaluated with a panel of synthetic phosphonates. Via 31P NMR spectroscopy, it is demonstrated that the GmPhnY* converts methylphosphonic acid to hydroxymethylphosphonic acid, which in turn is oxidized by GmPhnZ1 to produce formic acid and Pi. In contrast, 2-aminoethylphosphonic acid is not a substrate for GmPhnY* and is therefore not a substrate for this pathway. These results thus reveal a new metabolic fate for methylphosphonic acid.

Enolase is a glycolytic metalloenzyme involved in carbon metabolism. The advantage of targeting enolase lies in its essentiality in many biological processes such as cell wall formation and RNA turnover and as a plasminogen receptor. We initially used a DARTS assay to identify enolase as a target in Escherichia coli. The antibacterial activities of α-, β-, and γ-substituted seven-member ring tropolones were first evaluated against four strains representing a range of Gram-negative bacteria. We observed that the chemical properties and position of the substituents on the tropolone ring play an important role in the biological activity of the investigated compounds. Both α- and β-substituted phenyl derivatives of tropolone were the most active with minimum inhibitory concentrations in the range of 11-14 μg/mL. The potential inhibitory activity of the synthetic tropolones was further evaluated using an enolase inhibition assay, X-ray crystallography, and molecular docking simulations. The catalytic activity of enolase was effectively inhibited by both the naturally occurring β-thujaplicin and the α- and β-substituted phenyl derivatives of tropolones with IC50 values in range of 8-11 μM. Ligand binding parameters were assessed by isothermal titration calorimetry and differential scanning calorimetry techniques and agreed with the in vitro data. Our studies validate the antibacterial potential of tropolones with careful consideration of the position and character of chelating moieties for stronger interaction with metal ions and residues in the enolase active site.

Bempedoic acid has emerged as a potent inhibitor of ATP-citrate lyase (ACLY), a target for the reduction of LDL cholesterol (LDL-C). We review the impact of bempedoic acid treatment on lipoprotein metabolism and atherosclerosis in preclinical models and patients with hypercholesterolemia.The liver-specific activation of bempedoic acid inhibits ACLY, a key enzyme linking glucose catabolism to lipogenesis by catalyzing the formation of acetyl-CoA from mitochondrial-derived citrate for de novo synthesis of fatty acids and cholesterol. Adenosine monophosphate-activated protein kinase activation by bempedoic acid is not required for its lipid-regulating effects in vivo. Mendelian randomization of large human study cohorts has validated ACLY inhibition as a target for LDL-C lowering and atheroprotection. In rodents, bempedoic acid decreases plasma cholesterol and triglycerides, and prevents hepatic steatosis. In apolipoprotein E-deficient (Apoe) mice, LDL receptor-deficient (Ldlr) mice and LDLR-deficient miniature pigs, bempedoic acid reduces LDL-C and attenuates atherosclerosis. LDLR expression and activity are increased in primary human hepatocytes and in Apoe mouse liver treated with bempedoic acid suggesting a mechanism for LDL-C lowering, although additional pathways are likely involved. Phase 2 and 3 clinical trials revealed that bempedoic acid effectively lowers LDL-C as monotherapy, combined with ezetimibe, added to statin therapy and in statin-intolerant hypercholesterolemic patients. Treatment does not affect plasma concentrations of triglyceride or other lipoproteins.The LDL-C-lowering and attenuated atherosclerosis in animal models and reduced LDL-C in hypercholesterolemic patients has validated ACLY inhibition as a therapeutic strategy. Positive results from phase 3 long-term cardiovascular outcome trials in high-risk patients are required for bempedoic acid to be approved for prevention of atherosclerosis.

Although enoyl-CoA hydratase/isomerase superfamily proteins are functionally diverse and extremely abundant in microbial and higher organism’s genome, they still have been elusively annotated. The genome of Cupriavidus necator H16 contains at least 54 enoyl-CoA hydratase/isomerase superfamily proteins that might influence on polyhydroxyalkanoate synthesis, but most of them are uncharacterized. Among them, we first determined crystal structure of H16_B0756 at a 2.0 A resolution. The protein exhibits unique amino acid sequences compared to the other isoforms with identity lower than 36%. The structure of H16_B0756 forms a trimeric architecture and showed canonical disk-shape. Interestingly, H16_B0756 has only one glutamate residue at the active site while other enoyl-CoA hydratases have two nucleophilic glutamate at the catalytic site. We found that the active site conformation of H16_B0756 is quite similar to that of 1,2-epoxyphenylacetyl-CoA isomerase (PaaG) rather than those of other enoyl-CoA hydratases. In addition to the structural comparison, gene neighborhoods analysis suggested that H16_B0756 might function in the ring compound degradation.

Argininosuccinate lyase (ASL) participates in arginine synthesis through catalysing a reversible reaction in which argininosuccinate (AS) converts into arginine and fumarate. ASL from Mycobacterium tuberculosis is essential for its growth. In this work, the crystal structure of the apo form of MtbASL was determined and reveals a tetrameric structure that is essential for its activity since the active sites are formed by residues from three different monomers. Subsequently, we determined the crystal structure of MtbASL-sulfate complex, and the ligand mimics the negatively charged intermediate. The complex structure and mutagenesis studies indicate that residues S282 and H161 might act as a catalytic dyad. A major conformational change in the MtbASL-SO4 complex structure could be observed upon sulfate binding, and this movement facilitates the interaction between substrate and the residues involved in catalysis. A different conformational change in the C-terminal domain could be observed in the MtbASL-SO4 complex compared with that in other homologues. This difference may be responsible for the lower activity of MtbASL, which is related to the slow growth rate of M. tuberculosis. The C-terminal domain is a potential allosteric site upon inhibitor binding. The various conformational changes and the diversity of the sequence of the potential allosteric site across the homologues might provide clues for designing selective inhibitors against M. tuberculosis.

Gellan is a widely used microbial polysaccharide and one of the more effective ways to expand its application value would be to investigate the mechanism of gellan lyase and to produce gellan oligosaccharide. In this study, efficient gellan degrading bacteria were screened. One of the strains with high efficient gellan degradation capacity was labeled PE1. Through physiological and biochemical analysis of 16S rDNA, the species was identified as Pseudoalteromonas hodoensis. The optimum conditions for enzymatic activity and how it was affected by metal ions were determined, and the results showed that the lyase activities were much higher than those of previously reported (about 20 times). The gellan degradation products were determined by thin-layer chromatography and the oligosaccharides were determined by high-efficiency liquid chromatography to analyze the action site of lyase. This study laid a solid foundation which elucidates the production and application of gellan oligosaccharides. Research highlights ● High efficiency gellan lyase producing bacteria ● Optimization of reaction conditions for gellan degradation ● Oligosaccharides were detected by TLC and HPLC to speculate the lyase action sites.

Elucidation of the role of entropy in enzymatic reactions can be utilized in the de novo design of enzymes or protein engineering. Recently, the change in entropy in the enzyme-catalyzed chorismate mutase reaction brought about uncertainty as a result of an experiment using PchB, in which the measured change in entropy was comparable to the reported value for the uncatalyzed reaction in an aqueous solution, which contrasted with the general proposed entropy-driven mechanism. On the basis of a sufficient sample from quantum mechanics/molecular mechanics molecular dynamics simulations, the entropic effect in the PchB-catalyzed chorismate mutase reaction has been determined for the first time. Calculations suggest that the PchB-catalyzed chorismate mutase reaction is entropy-driven. An additional entropic penalty was revealed from the substrate preorganization process, which led to a remarkable apparent entropic effect in the reaction. Our findings provide an explanation of the uncertainty regarding the large measured entropic effect in experiments and confirm the entropy-driven character of the PchB-catalyzed chorismate mutase reaction. The clarification of the change in entropy in this enzymatic reaction would provide useful clues for future enzyme design projects.

Here, we report the first bacterial cytochrome P450, CYP154C8, that catalyzes the C-C bond cleavage reaction of steroids. A major change in product distribution is observed with CYP154C8, when the reactions are supported by NADPH and spinach redox partners ferredoxin and ferredoxin reductase, compared with previously reported reactions supported by NADH and redox partners containing putidaredoxin and putidaredoxin reductase. The NMR-based structural elucidation of reaction products reveals 21-hydroxyprednisone as the major product for prednisone, while the other product is identified as 1-dehydroadrenosterone obtained due to C-C bond cleavage. A similar pattern of product formation is observed with cortisone, hydrocortisone, and prednisone. The reaction catalyzed by CYP154C8 in the presence of oxygen surrogates also prominently shows the formation of C-C bond cleavage products.

Computational design with supersecondary structures as building blocks has proven effective in the construction of new proteins with controlled geometries. So far, this approach has primarily exploited amplification, effectively harnessing the internal folding propensity of self-compatible fragments to achieve sufficient enthalpy for folding. Here we exploit an interface-driven strategy to depart from the repeat design realm, constructing an asymmetric, globular domain from heterologous supersecondary structures. We report the successful design of a dRP lyase domain fold, which agrees with the experimental NMR structure at atomic accuracy (backbone RMSD of 0.94 A). Our results show that the residual folding information within conserved fragments, combined with efficient interface-directed sampling, can effectively yield globular proteins with novel sequences and biophysical properties.

Rhamnogalacturonan I (RG-I) is a domain of plant cell wall pectin. The rhamnogalacturonan lyase (RGL) enzyme (EC 4.2.2.23) degrades RG-I by cleaving the α-1,4 glycosidic bonds located between the l-rhamnose and d-galacturonic residues of the main chain. While RGL's biochemical mode of action is well known, its effects on plant physiology remain unclear. To investigate the role of the RGL enzyme in plants, we have expressed the Solyc11g011300 gene under a constitutive promoter (CaMV35S) in tomato cv. 'Ohio 8245' and evaluated the expression of this and other RGL genes, enzymatic activity and alterations in vegetative tissue, and tomato physiology in transformed lines compared to the positive control (plants harboring the pCAMBIA2301 vector) and the isogenic line. The highest expression levels of the Solyc11g011300, Solyc04g076630, and Solyc04g076660 genes were observed in leaves and roots and at 10 and 20 days after anthesis (DAA). Transgenic lines exhibited lower RGL activity in leaves and roots and during fruit ripening, whereas higher activity was observed at 10, 20, and 30 DAA than in the isogenic line and positive control. Both transgenic lines showed a lower number of seeds and fruits, higher root length, and less pollen germination percentage and viability. In red ripe tomatoes, transgenic fruits showed greater firmness, longer shelf life, and reduced shriveling than did the isogenic line. Additionally, a delay of one week in fruit ripening in transgenic fruits was also recorded. Altogether, our data demonstrate that the Solyc11g011300 gene participates in pollen tube germination, fruit firmness, and the fruit senescence phenomena that impact postharvest shelf life.

Several new molecules with different thio-scaffolds were designed, synthesised, and evaluated biologically as inhibitors of Carbonic Anhydrases (CAIs). The structure-activity relationship analysis identified thioether derivatives, here reported, as a potent and selective CAIs against hCA II and hCA IX. High resolution X-ray structure of inhibitor bound hCA II revealed extensive interactions with the hydrophobic pocket of active site and provided molecular insight into the binding properties of these new inhibitors.

The present study demonstrated atrazine detoxification by intracellular crude enzyme extracts of Pseudomonas spp. strains ACB and TLB. Indigenous bacterial protein-based remediation techniques could be an alternative to bioaugmentation which pose multiple challenges when applied to the field. Intracellular enzymes were extracted from strains ACB and TLB and their degradation potential of 10 mg L-1 was determined using Gas Chromatography; further, enzyme extracts were subjected to protein profiling studies. In span of 6 h, enzyme extracts of strain ACB showed maximum degradation at 30 °C and 40 °C (71%) and enzyme extracts of strain TLB showed maximum degradation at 40 °C (48%). Atrazine degradation by enzyme extracts of strain ACB showed maximum degradation at pH 7 (71%) and pH 6 (69%) in 6 h. Similarly, enzyme extracts of strain TLB showed maximal degradation at pH 6 (46%) in 6 h. The present study demonstrated, for the first time, efficient atrazine remediation by intracellular crude enzyme extracts from epiphytic root bacteria at a range of temperature and pH conditions. Protein profiling studies indicated that atrazine induced expression of CoA ester lyase and alkyl hydroperoxide reductase in the strains ACB and TLB respectively. Expressions of these proteins have never been associated with atrazine exposure.

Despite diversification into biochemically distinct enzyme families, rv0812 encodes a single active site that functions as an enzymatically bifunctional aminodeoxychorismate lyase (ADCL) and D–amino acid transaminase (DAAT) and may couple replication and division in Mycobacterium tuberculosis., Comparative sequence analysis has enabled the annotation of millions of genes from organisms across the evolutionary tree. However, this approach has inherently biased the annotation of phylogenetically ubiquitous, rather than species-specific, functions. The ecologically unusual pathogen Mycobacterium tuberculosis (Mtb) has evolved in humans as its sole reservoir and emerged as the leading bacterial cause of death worldwide. However, the physiological factors that define Mtb’s pathogenicity are poorly understood. Here, we report the structure and function of a protein that is required for optimal in vitro fitness and bears homology to two distinct enzymes, Rv0812. Despite diversification of related orthologues into biochemically distinct enzyme families, rv0812 encodes a single active site with aminodeoxychorismate lyase and D–amino acid transaminase activities. The mutual exclusivity of substrate occupancy in this active site mediates coupling between nucleic acid and cell wall biosynthesis, prioritizing PABA over D-Ala/D-Glu biosynthesis. This bifunctionality reveals a novel, enzymatically encoded fail-safe mechanism that may help Mtb and other bacteria couple replication and division.

S-adenosylmethionine lyase (SAMase) of bacteriophage T3 degrades the intracellular SAM pools of the host E. coli cells, thus inactivating a crucial metabolite involved in plethora of cellular functions, including DNA methylation. SAMase is the first viral protein expressed upon infection and its activity prevents methylation of the T3 genome. Maintenance of the phage genome in a fully unmethylated state has a profound effect on the infection strategy — it allows T3 to shift from a lytic infection under normal growth conditions to a transient lysogenic infection under glucose starvation. Using single-particle Cryo-EM and biochemical assays, we demonstrate that SAMase performs its function by not only degrading SAM, but also by interacting with and efficiently inhibiting the host’s methionine S-adenosyltransferase (MAT) — the enzyme that produces SAM. Specifically, SAMase triggers open-ended head-to-tail assembly of E. coli MAT into an unusual linear filamentous structure in which adjacent MAT tetramers are joined together by two SAMase dimers. Molecular dynamics simulations together with normal mode analyses suggest that the entrapment of MAT tetramers within filaments leads to an allosteric inhibition of MAT activity due to a shift to low-frequency high-amplitude active site-deforming modes. The amplification of uncorrelated motions between active site residues weakens MAT’s ability to withhold substrates, explaining the observed loss of function. We propose that the dual function of SAMase as an enzyme that degrades SAM and as an inhibitor of MAT activity has emerged to achieve an efficient depletion of the intracellular SAM pools.IMPORTANCESelf-assembly of enzymes into filamentous structures in response to specific metabolic cues has recently emerged as a widespread strategy of metabolic regulation. In many instances filamentation of metabolic enzymes occurs in response to starvation and leads to functional inactivation. Here, we report that bacteriophage T3 modulates the metabolism of the host E. coli cells by recruiting a similar strategy — silencing a central metabolic enzyme by subjecting it to phage-mediated polymerization. This observation points to an intriguing possibility that virus-induced polymerization of the host metabolic enzymes might be a common mechanism implemented by viruses to metabolically reprogram and subdue infected cells.

Sphingosine-1-phosphate lyase insufficiency syndrome (SPLIS) is a rare metabolic disorder caused by inactivating mutations in sphingosine-1-phosphate lyase 1 (SGPL1), which is required for the final step of sphingolipid metabolism. SPLIS features include steroid-resistant nephrotic syndrome and impairment of neurological, endocrine, and hematopoietic systems. Many affected individuals die within the first 2 years. No targeted therapy for SPLIS is available. We hypothesized that SGPL1 gene replacement would address the root cause of SPLIS, thereby serving as a universal treatment for the condition. As proof of concept, we evaluated the efficacy of adeno-associated virus 9-mediated transfer of human SGPL1 (AAV-SPL) given to newborn Sgpl1-KO mice that model SPLIS and die in the first weeks of life. Treatment dramatically prolonged survival and prevented nephrosis, neurodevelopmental delay, anemia, and hypercholesterolemia. STAT3 pathway activation and elevated proinflammatory and profibrogenic cytokines observed in KO kidneys were attenuated by treatment. Plasma and tissue sphingolipids were reduced in treated compared with untreated KO pups. SGPL1 expression and activity were measurable for at least 40 weeks. In summary, early AAV-SPL treatment prevents nephrosis, lipidosis, and neurological impairment in a mouse model of SPLIS. Our results suggest that SGPL1 gene replacement holds promise as a durable and universal targeted treatment for SPLIS.

Microbial phosphonate biosynthetic machinery has been identified in ~5% of bacterial genomes and encodes natural products like fosfomycin as well as cell surface decorations. Almost all biological phosphonates originate from the rearrangement of phosphoenolpyruvate (PEP) to phosphonopyruvate (PnPy) catalyzed by PEP mutase (Ppm), and PnPy is often converted to phosphonoacetaldehyde (PnAA) by PnPy decarboxylase (Ppd). Seven enzymes are known or likely to act on either PnPy or PnAA as early branch points en route to diverse biosynthetic outcomes, and these enzymes may be broadly classified into three reaction types: hydride transfer, aminotransfer, and carbon-carbon bond formation. However, the relative abundance of these branch points in microbial phosphonate biosynthesis is unknown. Also unknown is the proportion of ppm-containing gene neighborhoods encoding new branch point enzymes and potentially novel phosphonates. In this study we computationally sorted 434 ppm-containing gene neighborhoods based on these seven branch point enzymes. Unsurprisingly, the majority (56%) of these pathways encode for production of the common naturally occurring compound 2-aminoethylphosphonate (AEP) or a hydroxylated derivative. The next most abundant genetically encoded intermediates were phosphonoalanine (PnAla, 9.2%), 2-hydroxyethylphosphonate (HEP, 8.5%), and phosphonoacetate (PnAc, 6%). Significantly, about 13% of the gene neighborhoods could not be assigned to any of the seven branch points and may encode novel phosphonates. Sequence similarity network analysis revealed families of unusual gene neighborhoods including possible production of phosphonoacrylate and phosphonofructose, the apparent biosynthetic use of the C-P lyase operon, and a virus-encoded phosphonate. Overall, these results highlight the utility of branch point inventories to identify novel gene neighborhoods and guide future phosphonate discovery efforts.IMPACT STATEMENTMicrobially-produced phosphonates are relatively rare and underexplored but include medically and agriculturally important molecules like fosfomycin and phosphinothricin, respectively. Because a single enzyme called phosphoenolpyruvate mutase (Ppm) inititates almost all phosphonate production, the composition of the ‘gene neighborhood’ surrounding a Ppm-encoding gene can inform hypotheses regarding the chemical output of this chromosomal region. After the initial Ppm-catalyzed reaction there are only a limited set of subsequently acting enzymes, or ‘branch points’, to direct these early-stage phosphonates to alternate chemical fates. However, the relative abundance of different branch points – or the existence of new ones – has not been evaluated. This study provides just such a ‘branch point inventory’ to determine relative proportions of known branch points and assess the diversity within each branch point. Significantly, this study suggests that a significant proportion (~13%) of gene neighborhoods do not fit into known branch points and therefore may be fertile hunting grounds for new phosphonate biochemistry.Data SummarySupporting information is available at Scholars Portal Dataverse (https://dataverse.scholarsportal.info/) with DOI 10.5683/SP2/T33ZP6. This includes scripts and the network data for visualizing in BiG-SCAPE and Cytoscape.

The functional properties of alginates are dictated by the monomer composition and molecular weight distribution. Mannuronan C-5 epimerases determine the former by epimerizing β-D-mannuronic acid residues (M) into α-L-guluronic acid residues (G). The molecular weight is affected by alginate lyases, which cleave alginate chains through β-elimination. The reaction mechanisms for the epimerization and cleavage are similar and some enzymes can perform both. These dualistic enzymes share high sequence identity with mannuronan C-5 epimerases without lyase activity, and the mechanism behind their activity as well as the amino acids responsible for it are still unknown. In this study, we investigate mechanistic determinants of the bifunctional epimerase and lyase activity of AlgE7 from Azotobacter vinelandii. Based on sequence analyses, a range of AlgE7 variants were constructed and subjected to activity assays and product characterization by NMR. Our results show that the lyase activity of AlgE7 is regulated by the type of ion present: Calcium promotes it, whereas NaCl reduces it. By using defined poly-M and poly-MG substrates, the preferred cleavage sites of AlgE7 were found to be M↓XM and G↓XM, where X can be either M or G. By studying AlgE7 mutants, R148 was identified as an important residue for the lyase activity, and the point mutant R148G resulted in an enzyme with only epimerase activity. Based on the results obtained in the present study we suggest a unified catalytic reaction mechanism for both epimerase and lyase activities where H154 functions as the catalytic base and Y149 as the catalytic acid.ImportancePost-harvest valorisation and upgrading of algal constituents is a promising strategy in the development of a sustainable bioeconomy based on algal biomass. In this respect, alginate epimerases and lyases are valuable enzymes for tailoring of the functional properties of alginate, a polysaccharide extracted from brown seaweed with numerous applications in food, medicine and material industries. By providing a better understanding of the reaction mechanism and of how the two enzyme reactions can be altered by changes in reaction conditions, this study opens for further applications of bacterial epimerases and lyases in enzymatic tailoring of alginate polymers.

Hydrogen sulfide (H2S) has recently been recognized as a novel gaseous transmitter with several anti-inflammatory properties. The role of host- derived H2S in infections by Pseudomonas aeruginosa was investigated in clinical and mouse models. H2S concentrations and survival was assessed in septic patients with lung infection. Animal experiments using a model of severe systemic multidrug-resistant P. aeruginosa infection were performed using mice with a constitutive knock-out of cystathionine-γ lyase (Cse) gene (Cse-/-) and wild-type mice with a physiological expression (Cse+/+). Experiments were repeated in mice after a) treatment with cyclophosphamide; b) bone marrow transplantation (BMT) from a Cse+/+ donor; c) treatment with H2S synthesis inhibitor aminooxyacetic acid (ΑΟΑΑ) or propargylglycine (PAG) and d) H2S donor sodium thiosulfate (STS) or GYY3147. Bacterial loads and myeloperoxidase activity were measured in tissue samples. The expression of quorum sensing genes (QS) was determined in vivo and in vitro. Cytokine concentration was measured in serum and incubated splenocytes. Patients survivors at day 28 had significantly higher serum H2S compared to non-survivors. A cut- off point of 5.3 μΜ discriminated survivors with sensitivity 92.3%. Mortality after 28 days was 30.9% and 93.7% in patients with H2S higher and less than 5.3 μΜ (p = 7 x 10−6). In mice expression of Cse and application of STS afforded protection against infection with multidrug-resistant P. aeruginosa. Cyclophosphamide pretreatment eliminated the survival benefit of Cse+/+ mice, whereas BMT increased the survival of Cse-/- mice. Cse-/- mice had increased pathogen loads compared to Cse+/+ mice. Phagocytic activity of leukocytes from Cse-/- mice was reduced but was restored after H2S supplementation. An H2S dependent down- regulation of quorum sensing genes of P.aeruginosa could be demonstrated in vivo and in vitro. Endogenous H2S is a potential independent parameter correlating with the outcome of P. aeruginosa. H2S provides resistance to infection by MDR bacterial pathogens., Author summary Multidrug- resistant Pseudomonas (P.) aeruginosa is one of the most common causes of ventilator-associated pneumonia (VAP), which consist one of the most common causes of sepsis in hospitalized patients. The increasing antimicrobial resistance of P. aeruginosa necessitates the investigation of new treatment strategies. In this study we highlight host derived hydrogen sulfide (H2S), produced by cystathionine-γ-lyase (CSE), as a new protective mechanism against Pseudomonas aeruginosa infection. We have shown that serum H2S levels above 5.3μM can discriminate survivors of P.aeruginosa related sepsis with high sensitivity and is correlated with reduced mortality. We verified this finding in an animal model of CSE knockout mice. The protective role of endogenous H2S resides in the enhancement of neutrophil recruitment and the phagocytic activity of neutrophils, as well as by inhibiting the cell-to-cell communication system of P. aeruginosa (quorum sensing system), rendering it more vulnerable to phagocytosis.

Nitrogen fixing-cyanobacteria can significantly improve the economic feasibility of cyanobacterial production processes by eliminating the requirement for reduced nitrogen. Anabaena sp. ATCC 33047 is a marine, heterocyst forming, nitrogen fixing cyanobacteria with a very short doubling time of 3.8 h. We developed a comprehensive genome-scale metabolic (GSM) model, iAnC892, for this organism using annotations and content obtained from multiple databases. iAnC892 describes both the vegetative and heterocyst cell types found in the filaments of Anabaena sp. ATCC 33047. iAnC892 includes 953 unique reactions and accounts for the annotation of 892 genes. Comparison of iAnC892 reaction content with the GSM of Anabaena sp. PCC 7120 revealed that there are 109 reactions including uptake hydrogenase, pyruvate decarboxylase, and pyruvate-formate lyase unique to iAnC892. iAnC892 enabled the analysis of energy production pathways in the heterocyst by allowing the cell specific deactivation of light dependent electron transport chain and glucose-6-phosphate metabolizing pathways. The analysis revealed the importance of light dependent electron transport in generating ATP and NADPH at the required ratio for optimal N2 fixation. When used alongside the strain design algorithm, OptForce, iAnC892 recapitulated several of the experimentally successful genetic intervention strategies that over produced valerolactam and caprolactam precursors.

It has been recognized for >50 years that cytochrome b5 (b5) stimulates some cytochrome P450 (P450)–catalyzed oxidations, but the basis of this function is still not understood well. The strongest stimulation of catalytic activity by b5 is in the P450 17A1 lyase reaction, an essential step in androgen synthesis from 21-carbon (C21) steroids, making this an excellent model system to interrogate b5 function. One of the issues in studying b5–P450 interactions has been the limited solution assay methods. We constructed a fluorescently labeled variant of human b5 that can be used in titrations. The labeled b5 bound to WT P450 17A1 with a Kd of 2.5 nM and rapid kinetics, on the order of 1 s−1. Only weak binding was observed with the clinical P450 17A1 variants E305G, R347H, and R358Q; these mutants are deficient in lyase activity, which has been hypothesized to be due to attenuated b5 binding. Kd values were not affected by the presence of P450 17A1 substrates. A peptide containing the P450 17A1 Arg-347/Arg-358 region attenuated Alexa 488-T70C-b5 fluorescence at higher concentrations. The addition of NADPH–P450 reductase (POR) to an Alexa 488-T70C-b5:P450 17A1 complex resulted in a concentration-dependent partial restoration of b5 fluorescence, indicative of a ternary P450:b5:POR complex, which was also supported by gel filtration experiments. Overall, these results are interpreted in the context of a dynamic and tight P450 17A1:b5 complex that also binds POR to form a catalytically competent ternary complex, and variants that disrupt this interaction have low catalytic activity.

Welan gum, a kind of microbial exopolysaccharides, produced by the genus Sphingomonas, have great potential for application in many fields, such as the food industry, cement production, and enhanced oil recovery. But there are still challenges to reduce the cost, enhance the production and the quality. Herein, the bioinformatics analysis of WelR gene was preformed, and the characterization and function of WelR, welan gum lyase, from Sphingomonas sp. WG were investigated for the first time. The results indicated that 382nd (Asn), 383rd (Met), 494th (Asn), and 568th (Glu) were the key amino acid residues, and C-terminal amino acids were essential to keeping the stability of WelR. The optimal temperature and pH of the enzymatic activity were found to be 25°C and 7.4, respectively. And WelR was good low temperature resistance and alkali resistant. K+, Mg2+, Ca2+, Mn2+, and EDTA increased WelR activities, in contrast to Zn2+. Coupled with the change in glucose concentration and growth profile, the qRT-PCR results indicated that WelR may degrade welan gum existing in the culture to maintain bacterial metabolism when glucose was depleted. This work will lay a theoretical foundation to establish new strategies for the regulation of welan gum biosynthesis.

Alginate is an anionic polysaccharide abundantly present in the cell walls of brown macroalgae. The enzymatic depolymerization is performed solely by alginate lyases (EC 4.2.2.x), categorized as polysaccharide lyases (PLs) belonging to 12 different PL families. Until now, the vast majority of the alginate lyases have been found in bacteria. We report here the first extensive characterization of four alginate lyases from a marine fungus, the ascomycete Paradendryphiella salina, a known saprophyte of seaweeds. We have identified four polysaccharide lyase encoding genes bioinformatically in P. salina, one PL8 (PsMan8A), and three PL7 alginate lyases (PsAlg7A, -B, and -C). PsMan8A was demonstrated to exert exo-action on polymannuronic acid, and no action on alginate, indicating that this enzyme is most likely an exo-acting polymannuronic acid specific lyase. This enzyme is the first alginate lyase assigned to PL8 and polymannuronic acid thus represents a new substrate specificity in this family. The PL7 lyases (PsAlg7A, -B, and -C) were found to be endo-acting alginate lyases with different activity optima, substrate affinities, and product profiles. PsAlg7A and PsMan8A showed a clear synergistic action for the complete depolymerization of polyM at pH 5. PsAlg7A depolymerized polyM to mainly DP5 and DP3 oligomers and PsMan8A to dimers and monosaccharides. PsAlg7B and PsAlg7C showed substrate affinities towards both polyM and polyG at pH 8, depolymerizing both substrates to DP9-DP2 oligomers. The findings elucidate how P. salina accomplishes alginate depolymerization and provide insight into an efficient synergistic cooperation that may provide a new foundation for enzyme selection for alginate degradation in seaweed bioprocessing.

Lytic enzymes play an essential role in the remodeling of bacterial peptidoglycan (PG), an extracellular mesh-like structure that retains the membrane in the context of high internal osmotic pressure. Peptidoglycan must be unfailingly stable to preserve cell integrity, but must also be dynamically remodeled for the cell to grow, divide, and insert macromolecular machines. The flagellum is one such macromolecular machine that transits the PG, and flagellar insertion is aided by localized activity of a dedicated PG lyase in Gram-negative bacteria. To date, there is no known dedicated lyase in Gram-positive bacteria for the insertion of flagella. Here, we take a reverse-genetic candidate-gene approach and find that cells mutated for the lytic transglycosylase CwlQ exhibit a severe defect in flagellum-dependent swarming motility. We further show that CwlQ is expressed by the motility sigma factor SigD and is secreted by the type III secretion system housed inside the flagellum. Nonetheless, cells with mutations of CwlQ remain proficient for flagellar biosynthesis even when mutated in combination with four other lyases related to motility (LytC, LytD, LytF, and CwlO). The PG lyase (or lyases) essential for flagellar synthesis in B. subtilis, if any, remains unknown. IMPORTANCE Bacteria are surrounded by a wall of peptidoglycan and early work in Bacillus subtilis was the first to suggest that bacteria needed to enzymatically remodel the wall to permit insertion of the flagellum. No PG remodeling enzyme alone or in combination, however, has been found to be essential for flagellar assembly in B. subtilis. Here, we take a reverse-genetic candidate-gene approach and find that the PG lytic transglycosylase CwlQ is required for swarming motility. Subsequent characterization determined that while CwlQ was coexpressed with motility genes and is secreted by the flagellar secretion apparatus, it was not required for flagellar synthesis. The PG lyase needed for flagellar assembly in B. subtilis remains unknown.

We report here on the salient role of protein mobility in accessing conformational landscapes that enable efficient enzyme catalysis. We are focused on yeast enolase, a highly conserved lyase with a TIM barrel domain and catalytic loop, as part of a larger study of the relationship of site selective protein motions to chemical reactivity within superfamilies. Enthalpically hindered variants were developed by replacement of a conserved hydrophobic side chain (Leu 343) with smaller side chains. Leu343 is proximal to the active site base in enolase, and comparative pH rate profiles for the valine and alanine variants indicate a role for side chain hydrophobicity in tuning the pKa of the catalytic base. However, the magnitude of a substrate deuterium isotope effect is almost identical for wild-type (WT) and Leu343Ala, supporting an unchanged rate-determining proton abstraction step. The introduced hydrophobic side chains at position 343 lead to a discontinuous break in both activity and activation energy as a function of side chain volume. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments were performed as a function of time and temperature for WT and Leu343Ala, and provide a spatially resolved map of changes in protein flexibility following mutation. Impacts on protein flexibility are localized to specific networks that arise at the protein-solvent interface and terminate in a loop that has been shown by X-ray crystallography to close over the active site. These interrelated effects are discussed in the context of long-range, solvent-accessible and thermally activated networks that play key roles in tuning the precise distances and interactions among reactants.

A library of 23 synthetic heparan sulfate (HS) oligosaccharides, varying in chain length, types, and positions of modifications, was used to analyze the substrate specificities of heparin lyase III enzymes from both Flavobacterium heparinum and Bacteroides eggerthii. The influence of specific modifications, including N-substitution, 2-O sulfation, 6-O sulfation, and 3-O sulfation on lyase III digestion was examined systematically. It was demonstrated that lyase III from both sources can completely digest oligosaccharides lacking O-sulfates. 2-O Sulfation completely blocked cleavage at the corresponding site; 6-O and 3-O sulfation on glucosamine residues inhibited enzyme activity. We also observed that there are differences in substrate specificities between the two lyase III enzymes for highly sulfated oligosaccharides. These findings will facilitate obtaining and analyzing the functional sulfated domains from large HS polymer, to better understand their structure/function relationships in biological processes.

Alginate oligosaccharides (AOS), belonging to the class of functional marine oligosaccharides, are low-molecular polymers linked by β-1,4-mannuronic acid (M) and α-1,4-guluronic acid (G), which could be classically obtained by enzymatic hydrolysis of alginate. With low viscosity and good water solubility, as well as anti-oxidant, immune regulation, anti-bacterial and anti-inflammatory activities, AOS have been widely used in medical science and functional food, green agriculture and other fields. As new bio-feed additives, AOS have broad potential applications in animal husbandry. In this review, the sources of alginate, chemical structure and preparation methods of AOS, and their biological activities and application in livestock and poultry are summarized. We expect this review could contribute to lay a foundation of application and further research for AOS in livestock and poultry.

Copper (Cu) is an essential element, however it's excess into the environment causes detrimental effect on plant and risks for public health. Four Cu and drought tolerant 1-aminocyclopropane-1-carboxylate (ACC) deaminase producing rhizobacteria were isolated from the roots of Trifolium repens L. growing on Cu smelter contaminated soils, characterized and identified based on 16S rRNA gene sequencing. A consortium of high ACC deaminase (53.74 μM α-ketobutyrate mg−1 protein h−1) producing bacteria Pseudomonas sp. strain TR15a + siderophore producing Bacillus aerophilus strain TR15c significantly (p < 0.05) produced better results for multiple-metal tolerance including Cu (1750 mg kg−1), antibiotic resistance (ampicillin, kanamycin, chloramphenicol, penicillin, tetracycline, and streptomycin) and plant growth promoting attributes (phosphate solubilization: 315 mg L−1, indole-3-acetic acid (IAA) production: 8 mg L−1, ammonia and hydrogen cyanide production) as compared to individual isolates. Pot scale experiment (enriched with 100 mg Cu kg−1) showed inoculation of Helianthus annuus seeds with consortium of TR15a + TR15c had significantly (p < 0.05) improved seed germination by 32%, total dry biomass by 64%, root Cu by 47% and shoot Cu by 75% as compared to uninoculated control whereas 0.2−7 fold higher results were observed for above stated parameters as compared to four individual isolates studied. The result suggests consortium of ACC deaminase producing Pseudomonas sp. TR15a and siderophore producing B. aerophilus TR15c could play a vital role in enhanced Cu uptake and improvement of biomass and may provide a better alternative for decontamination of Cu contaminated natural ecosystem than individual isolates. © 2021 Elsevier Ltd. The authors acknowledge the work support by RFBR, Russia (Project No. 19-516-45006) and DST, India (INT/ RUS / RFBR /363) and the Ministry of Science and Higher Education of the Russian Federation (agreement No. 02.A03.21.0006). We also thank Prof. Katarzyna Turnau and Rafal Wazny, Department of Bioremediation, Malopolska Center of Biotechnology, University of Jagiellonian, Krakow for giving assistance in identification of isolated rhizobacteria.