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A novel lactonase from Mycoplasma synoviae 53 (MS53-0025) and Mycoplasma agalactiae PG2 (MAG-6390) was characterized by protein structure determination, molecular docking, gene context analysis, and library screening. The crystal structure of MS53-0025 was

The exterior surface of the human pathogen Campylobacter jejuni is coated with a capsular polysaccharide (CPS) that consists of a repeating sequence of 2-5 different sugars that can be modified with various molecular decorations. In the HS:2 serotype from strain NCTC 11168, the repeating unit within the CPS is composed of d-ribose, N -acetyl-d-galactosamine, and a d-glucuronic acid that is further amidated with either serinol or ethanolamine. The d-glucuronic acid moiety is also decorated with d-glycero-l-gluco-heptose. Here, we show that two different GT2 glycosyltransferases catalyze the transfer of N -acetyl-d-galactosamine from UDP-NAc-d-galactosamine furanoside to the C4-hydroxyl group of the d-glucuronamide moiety at the growing end of the capsular polysaccharide chain. Catalytic activity was not observed with glycosides of d-glucuronic acid, and thus, the C6-carboxylate of the d-glucuronic acid moiety must be amidated prior to chain elongation. One of these enzymes comprises the N-terminal domain of Cj1438 (residues 1-325) and the other is from the N-terminal domain of Cj1434 (residues 1-327). These two glycosyltransferases are ∼87% identical in sequence, but it is not clear why there are two glycosyltransferases from the same gene cluster that apparently catalyze the same reaction. This discovery represents the second polymerizing glycosyltransferase that has been isolated and functionally characterized for the biosynthesis of the capsular polysaccharide in the HS:2 serotype of C. jejuni .

Campylobacter jejuni is the leading cause of food poisoning in Europe and North America. The exterior surface of this bacterium is encased by a capsular polysaccharide that is attached to a diacyl glycerol phosphate anchor via a poly-Kdo (3-deoxy-d- manno -oct-2-ulosinic acid) linker. In the HS:2 serotype of C. jejuni NCTC 11168, the repeating trisaccharide consists of d-ribose, N -acetyl-d-glucosamine, and d-glucuronate. Here, we show that the N-terminal domain of Cj1432 (residues 1-356) is responsible for the reaction of the C2 hydroxyl group from the terminal d-ribose moiety of the growing polysaccharide chain with UDP-d-glucuronate as the donor substrate. This discovery represents the first biochemical identification and functional characterization of a glycosyltransferase responsible for the polymerization of the capsular polysaccharide of C. jejuni . The product of the reaction catalyzed by the N-terminal domain of Cj1432 is the substrate for the reaction catalyzed by the C-terminal domain of Cj1438 (residues 453-776). This enzyme catalyzes amide bond formation using the C6 carboxylate of the terminal d-glucuronate moiety and ( S )-serinol phosphate as substrates. It is also shown that Cj1435 catalyzes the hydrolysis of phosphate from the product catalyzed by the C-terminal domain of Cj1438. These results demonstrate that amide decoration of the d-glucuronate moiety occurs after the incorporation of this sugar into the growing polysaccharide chain.

This study aimed to elucidate the mechanism of Huachansu Injection(HCSI) against colorectal cancer(CRC) using network pharmacology, molecular docking technology, and cellular experimental. This research group initially used LC-MS/MS to detect the content of 16 bufadienolides in HCSI. Ten bufadienolide components were selected based on a content threshold of greater than 10 ng·mL~(-1). Their potential targets were further predicted using the SwissTargetPrediction database. CRC-related targets were obtained through GeneCards, OMIM, TTD, and PharmGKB databases. The intersection targets of HCSI in the treatment of CRC were obtained through Venny. The "active component-target-disease" network and target protein-protein interaction(PPI) network were constructed via Cytoscape software. Core targets were screened based on the degree values. Gene Ontology(GO) function and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analyses were performed on these key targets. Molecular docking was conducted using AutoDock software on major bufadienolide active components and key targets. Different concentrations of HCSI, psi-bufarenogin(BUF), and bufotalin(BFT) were tested for their effects on cell viability, migration, and apoptosis rates in CRC HCT116 cells. Western blot was conducted to detect the expression of proteins related to the PI3K/Akt/mTOR signaling pathway in HCT116 cells. Eight main active components of HCSI, including arenobufagin, BUF, and BFT, as well as 20 key targets of HCSI in combating CRC, such as EGFR, IL6, and mTOR, were identified. Based on KEGG pathway enrichment and molecular docking results, the PI3K/Akt/mTOR signaling pathway was selected for further verification. Cellular experimental demonstrated that HCSI, BUF, and BFT significantly inhibited the proliferation and migration abilities of HCT116 cells, induced apoptosis in these cells, and downregulated the expression of PI3K/Akt/mTOR pathway-related proteins. This result suggests that HCSI, BUF, and BFT may exert their anti-CRC effects by regulating the PI3K/Akt/mTOR signaling pathway through targets such as mTOR and PIK3CA. This study provides theoretical evidence for exploring the active ingredients and mechanism of HCSI against CRC.

Campylobacter jejuni is the leading cause of food poisoning in North America. The exterior surface of this bacterium is coated with a capsular polysaccharide (CPS) that consists of a repeating sequence of 2-5 different carbohydrates that is anchored to the outer membrane. Heptoses of various configurations are among the most common monosaccharides that have been identified within the CPS. It is currently thought that all heptose variations derive from the modification of GDP-d- glycero -α-d- manno -heptose (GMH). From the associated gene clusters for CPS biosynthesis, we have identified 20 unique enzymes with different substrate profiles that are used by the various strains and serotypes of C. jejuni to make six different stereoisomers of GDP-6-deoxy-heptose, four stereoisomers of GDP-d- glycero -heptoses, and two stereoisomers of GDP-3,6-dideoxy-heptoses starting from d-sedoheptulose-7-phosphate. The modification enzymes include a C4-dehydrogenase, a C4,6-dehydratase, three C3- and/or C5-epimerases, a C3-dehydratase, eight C4-reductases, two pyranose/furanose mutases, and four enzymes for the formation of GMH from d-sedoheptulose-7-phosphate. We have mixed these enzymes in different combinations to make novel GDP-heptose modifications, including GDP-6-hydroxy-heptoses, GDP-3-deoxy-heptoses, and GDP-3,6-dideoxy-heptoses.

Campylobacter jejuni is the leading cause of food poisoning in the United States. Surrounding the exterior surface of this bacterium is a capsular polysaccharide (CPS) that helps protect the organism from the host immune system. The CPS is composed of a repeating sequence of common and unusual sugar residues, including relatively rare heptoses. In the HS:5 serotype, we identified four enzymes required for the biosynthesis of GDP-3,6-dideoxy-β-l- ribo -heptose. In the first step, GDP-d- glycero -α-d- manno -heptose is dehydrated to form GDP-6-deoxy-4-keto-α-d- lyxo -heptose. This product is then dehydrated by a pyridoxal phosphate-dependent C3-dehydratase to form GDP-3,6-dideoxy-4-keto-α-d- threo -heptose before being epimerized at C5 to generate GDP-3,6-dideoxy-4-keto-β-l- erythro -heptose. In the final step, a C4-reductase uses NADPH to convert this product to GDP-3,6-dideoxy-β-l- ribo -heptose. These results are at variance with the previous report of 3,6-dideoxy-d- ribo -heptose in the CPS from serotype HS:5 of C. jejuni . We also demonstrated that GDP-3,6-dideoxy-β-l- xylo -heptose is formed using the corresponding enzymes found in the gene cluster from serotype HS:11 of C. jejuni . The utilization of different C4-reductases from other serotypes of C. jejuni enabled the formation of GDP-3,6-dideoxy-α-d- arabino -heptose and GDP-3,6-dideoxy-α-d- lyxo -heptose.

Campylobacter jejuni is a human pathogen and the leading cause of food poisoning in the United States and Europe. Surrounding the exterior surface of this bacterium is a capsular polysaccharide (CPS) that consists of a repeating sequence of common and unusual carbohydrate segments. At least 10 different heptose sugars have thus far been identified in the various strains of C. jejuni . The accepted biosynthetic pathway for the construction of the 6-deoxy-heptoses begins with the 4,6-dehydration of GDP-d- glycero -d- manno -heptose by a dehydratase, followed by an epimerase that racemizes C3 and/or C5 of the product GDP-6-deoxy-4-keto-d- lyxo -heptose. In the final step, a C4-reductase catalyzes the NADPH reduction of the resulting 4-keto product. However, in some strains and serotypes of C. jejuni , there are two separate C4-reductases with different product specificities in the gene cluster for CPS formation. Five pairs of these tandem C4-reductases were isolated, and the catalytic properties were ascertained. In four out of five cases, one of the two C4-reductases is able to catalyze the isomerization of C3 and C5 of GDP-6-deoxy-4-keto-d- lyxo -heptose, in addition to the catalysis of the reduction of C4, thus bypassing the requirement for a separate C3/C5-isomerase. In each case, the 3'-end of the gene for the first C4-reductase contains a poly-G tract of 8-10 guanine residues that may be used to control the expression and/or catalytic activity of either C4-reductase. The three-dimensional structure of the C4-reductase from serotype HS:15, which only does a reduction of C4, was determined to 1.45 Å resolution in the presence of NADPH and GDP.

Campylobacter jejuni is the leading cause of food poisoning in the United States and Europe. A capsular polysaccharide that coats the exterior of the bacterium helps evade the host immune system. At least 33 different strains of C. jejuni have been identified, and the chemical structures of 12 different capsular polysaccharides (CPSs) have been characterized from various serotypes. Thus far, 10 different heptose sugars have been found in the chemically characterized CPSs, and each of these are currently thought to originate from the modification of GDP-d- glycero -d- manno -heptose by the successive action of 4,6-dehydratase (or C4-dehydrogenase), C3- or C3/C5-epimerase, and C4-reductase. Within the sequenced strains of C. jejuni , we have identified 25 different C4-reductases that cluster into nine groups at a sequence identity of >90%. Eight of the proteins from seven different clusters were purified, and their product profiles were determined with GDP-6-deoxy-4-keto-heptose substrates using NMR and ESI mass spectrometry. The isolated products included GDP-6-deoxy-l- gluco -heptose (serotype HS:2), GDP-6-deoxy-l- galacto -heptose (serotype HS:42), GDP-6-deoxy-l- gulo -heptose (serotype HS:15), GDP-6-deoxy-d- ido -heptose (serotypes HS:3, HS:4, and HS:33), GDP-6-deoxy-d- manno -heptose (serotype HS:53), and GDP-6-deoxy-d- altro -heptose (serotype HS:23/36). Based on these observations, the product specificity can be reliably predicted for 14 additional C4-reductases from C. jejuni . The remaining three C4-reductases are highly likely to be required for the biosynthesis of 3,6-dideoxy-heptose products.

Campylobacter jejuni is a human pathogen and one of the leading causes of food poisoning in Europe and the United States. The outside of the bacterium is coated with a capsular polysaccharide that assists in the evasion of the host immune system. Many of the serotyped strains of C. jejuni contain a 6-deoxy-heptose moiety that is biosynthesized from GDP-d- glycero -d- manno -heptose by the successive actions of a 4,6-dehydratase, a C3/C5-epimerase, and a C4-reductase. We identified 18 different C3/C5-epimerases that could be clustered together into three groups at a sequence identity of >89%. Four of the enzymes from the largest cluster (from serotypes HS:3, HS:10, HS:23/36, and HS:41) were shown to only catalyze the epimerization at C3. Three enzymes from the second largest cluster (HS:2, HS:15, and HS:42) were shown to catalyze the epimerization at C3 and C5. Enzymes from the third cluster were not characterized. The three-dimensional structures of the epimerases from serotypes HS:3, HS:23/36, HS:15, and HS:41 were determined to resolutions of 1.5-1.9 Å. The overall subunit architecture places these enzymes into the diverse "cupin" superfamily. Within X-ray coordinate error, the immediate regions surrounding the active sites are identical, suggesting that factors extending farther out may influence product outcome. The X-ray crystal structures are consistent with His-67 and Tyr-134 acting as general acid/base catalysts for the epimerization of C3 and/or C5. Two amino acid changes (A76V/C136L) were enough to convert the C3-epimerase from serotype HS:3 to one that could now catalyze the epimerization at both C3 and C5.

Plexin-domain containing 2 (PLXDC2) has been reported as an oncoprotein in several human malignancies. However, its expression and roles in gastric cancer remain largely unclear. In this study, we found that PLXDC2 was highly expressed in gastric cancer tissues, and the expression levels were positively correlated with clinicopathological features, but negatively with the patients' outcome. Cox regression analysis identified PLXDC2 as an independent prognostic indicator for the patients. Knockdown of PLXDC2 markedly suppressed the in vitro invasion and in vivo metastasis of gastric cancer cells, while overexpression of PLXDC2 resulted in opposite effects. Mechanistically, PLXDC2 enhanced the level of phosphorylated Cortactin (p-Cortactin) by physically interacting with protein tyrosine phosphatase 1B (PTP1B), an important dephosphorylase, to prevent its dephosphorylating of p-Cortactin, thereby promoting the formation of invadopodia. Collectively, our results indicate that PLXDC2 contributes to the invasion and metastasis of gastric cancer by inhibiting PTP1B to facilitate the invadopodium formation, and may serve as a potential prognostic biomarker and a therapeutic target for this disease., (© 2022. The Author(s).)

Campylobacter jejuni is a human pathogen and a leading cause of food poisoning in the United States and Europe. Surrounding the outside of the bacterium is a carbohydrate coat known as the capsular polysaccharide. Various strains of C. jejuni have different sequences of unusual sugars and an assortment of decorations. Many of the serotypes have heptoses with differing stereochemical arrangements at C2 through C6. One of the many common modifications is a 6-deoxy-heptose that is formed by dehydration of GDP-d-glycero-α-d-manno-heptose to GDP-6-deoxy-4-keto-d-lyxo-heptose via the action of the enzyme GDP-d-glycero-α-d-manno-heptose 4,6-dehydratase. Herein, we report the biochemical and structural characterization of this enzyme from C. jejuni 81-176 (serotype HS:23/36). The enzyme was purified to homogeneity, and its three-dimensional structure was determined to a resolution of 2.1 Å. Kinetic analyses suggest that the reaction mechanism proceeds through the formation of a 4-keto intermediate followed by the loss of water from C5/C6. Based on the three-dimensional structure, it is proposed that oxidation of C4 is assisted by proton transfer from the hydroxyl group to the phenolate of Tyr-159 and hydride transfer to the tightly bound NAD + in the active site. Elimination of water at C5/C6 is most likely assisted by abstraction of the proton at C5 by Glu-136 and subsequent proton transfer to the hydroxyl at C6 via Ser-134 and Tyr-159. A bioinformatic analysis identified 19 additional 4,6-dehydratases from serotyped strains of C. jejuni that are 89-98% identical in the amino acid sequence, indicating that each of these strains should contain a 6-deoxy-heptose within their capsular polysaccharides.

Non-typhoidal Salmonella are capable of colonizing livestock and humans, where they can progressively cause disease. Previously, a library of targeted single-gene deletion mutants of Salmonella enterica serotype Typhimurium was inoculated to ligated ileal loops in calves to identify genes under selection. Of those genes identified, a cluster of genes is related to carbohydrate metabolism and transportation. It is proposed that an incoming carbohydrate is first phosphorylated by a phosphoenolpyruvate-dependent phosphotransferase system. The metabolite is further phosphorylated by the kinase STM3781 and then cleaved by the aldolase STM3780. STM3780 is functionally annotated as a class II fructose-bisphosphate aldolase. The aldolase was purified to homogeneity, and its aldol condensation activity with a range of aldehydes was determined. In the condensation reaction, STM3780 was shown to catalyze the abstraction of the pro- S hydrogen from C3 of dihydroxyacetone and subsequent formation of a carbon-carbon bond with S stereochemistry at C3 and R stereochemistry at C4. The best aldehyde substrate was identified as l-threouronate. Surprisingly, STM3780 was also shown to catalyze the condensation of two molecules of dihydroxyacetone phosphate to form the branched carbohydrate dendroketose bisphosphate.

The phosphotriesterase from Sphingobium sp. TCM1 ( Sb -PTE) is notable for its ability to hydrolyze a broad spectrum of organophosphate triesters, including organophosphorus flame retardants and plasticizers such as triphenyl phosphate and tris(2-chloroethyl) phosphate that are not substrates for other enzymes. This enzyme is also capable of hydrolyzing any one of the three ester groups attached to the central phosphorus core. The enantiomeric isomers of 1,1'-bi-2-naphthol (BINOL) have become among the most widely used chiral auxiliaries for the chemical synthesis of chiral carbon centers. PTE was tested for its ability to hydrolyze a series of biaryl phosphate esters, including mono- and bis-phosphorylated BINOL derivatives and cyclic phosphate triesters. Sb -PTE was shown to be able to catalyze the hydrolysis of the chiral phosphate triesters with significant stereoselectivity. The catalytic efficiency, k cat / K m , of Sb -PTE toward the test phosphate triesters ranged from ∼10 to 10 5 M -1 s -1 . The product ratios and stereoselectivities were determined for four pairs of phosphorylated BINOL derivatives.

Objective: To investigate the pathological characteristics and the clinical significance of novel coronavirus (2019-nCoV)-infected pneumonia (termed by WHO as coronavirus disease 2019, COVID-19). Methods: Minimally invasive autopsies from lung, heart, kidney, spleen, bone marrow, liver, pancreas, stomach, intestine, thyroid and skin were performed on three patients died of novel coronavirus pneumonia in Chongqing, China. Hematoxylin and eosin staining (HE), transmission electron microcopy, and histochemical staining were performed to investigate the pathological changes of indicated organs or tissues. Immunohistochemical staining was conducted to evaluate the infiltration of immune cells as well as the expression of 2019-nCoV proteins. Real time PCR was carried out to detect the RNA of 2019-nCoV. Results: Various damages were observed in the alveolar structure, with minor serous exudation and fibrin exudation. Hyaline membrane formation was observed in some alveoli. The infiltrated immune cells in alveoli were majorly macrophages and monocytes. Moderate multinucleated giant cells, minimal lymphocytes, eosinophils and neutrophils were also observed. Most of infiltrated lymphocytes were CD4-positive T cells. Significant proliferation of type Ⅱ alveolar epithelia and focal desquamation of alveolar epithelia were also indicated. The blood vessels of alveolar septum were congested, edematous and widened, with modest infiltration of monocytes and lymphocytes. Hyaline thrombi were found in a minority of microvessels. Focal hemorrhage in lung tissue, organization of exudates in some alveolar cavities, and pulmonary interstitial fibrosis were observed. Part of the bronchial epithelia were exfoliated. Coronavirus particles in bronchial mucosal epithelia and type Ⅱ alveolar epithelia were observed under electron microscope. Immunohistochemical staining showed that part of the alveolar epithelia and macrophages were positive for 2019-nCoV antigen. Real time PCR analyses identified positive signals for 2019-nCoV nucleic acid. Decreased numbers of lymphocyte, cell degeneration and necrosis were observed in spleen. Furthermore, degeneration and necrosis of parenchymal cells, formation of hyaline thrombus in small vessels, and pathological changes of chronic diseases were observed in other organs and tissues, while no evidence of coronavirus infection was observed in these organs. Conclusions: The lungs from novel coronavirus pneumonia patients manifest significant pathological lesions, including the alveolar exudative inflammation and interstitial inflammation, alveolar epithelium proliferation and hyaline membrane formation. While the 2019-nCoV is mainly distributed in lung, the infection also involves in the damages of heart, vessels, liver, kidney and other organs. Further studies are warranted to investigate the mechanism underlying pathological changes of this disease.

Organophosphate flame retardants are used to inhibit combustion and increase plasticity in plastics and durable foams. While not neurotoxic, these compounds are potential carcinogens, endocrine disrupters, and developmental toxins. The phosphotriesterase from Sphingobium sp. TCM1 ( Sb -PTE) is unique among phosphotriesterase enzymes for its ability to hydrolyze these compounds and its ability to hydrolyze any one of the three different ester bonds within a given substrate. In some cases, the extent of hydrolysis of a methyl ester exceeds that of a p -nitrophenyl ester within a single substrate. There is a stereochemical component to this hydrolysis where the two enantiomers of chiral substrates give different product ratios. To investigate the stereoselectivity for the product distribution of Sb -PTE, a series of 24 phosphotriesters were synthesized with all possible combinations of methyl, cyclohexyl, phenyl, and p -nitrophenyl esters. Prochiral compounds were made chiral by differential isotopic labeling using a chemo/enzymatic strategy, which allowed the differentiation of hydrolysis for each ester in all but two compounds. The rate equations for this unique enzymatic mechanism were derived; the product ratios were determined for each substrate, and the individual kinetic constants for hydrolysis of each ester within each substrate were measured. The findings are consistent with the rate-limiting step for substrate hydrolysis catalyzed by Sb -PTE being the formation of a phosphorane-like intermediate and the kinetic constants and product ratios being dictated by a combination of transition state energies, inductive effects, and stereochemical constraints.

Nucleoside analogues are among the most common medications given for the treatment of viral infections and cancers. The therapeutic effectiveness of nucleoside analogues can be dramatically improved by phosphorylation. The ProTide approach was developed using a phosphorylated nucleoside that is masked by esterification with an amino acid and phenol forming a chiral phosphorus center. The biological activity of the ProTides depends, in part, on the stereochemistry at phosphorus, and thus, it is imperative that efficient methods be developed for the chemical synthesis and isolation of diastereomerically pure ProTides. Chiral ProTides are often synthesized by direct displacement of a labile phenol ( p -nitrophenol or pentafluorophenol) from a chiral phosphoramidate precursor with the appropriate nucleoside analogue. The ability to produce these chiral products is dictated by the synthesis of the chiral phosphoramidate precursors. The enzyme phosphotriesterase (PTE) from Pseudomonas diminuta is well-known for its high stereoselectivity and broad substrate profile. Screening PTE variants from enzyme evolution libraries enabled the identification of variants of PTE that can stereoselectively hydrolyze the chiral phosphoramidate precursors. The variant G60A-PTE exhibits a 165-fold preference for hydrolysis of the R P isomer, while the variant In1W-PTE has a 1400-fold preference for hydrolysis of the S P isomer. Using these mutants of PTE, the S P and R P isomers were isolated on a preparative scale with no detectable contamination of the opposite isomer. Combining the simplicity of the enzymatic resolution of the precursor with the latest synthetic strategy will facilitate the production of diastereometrically pure nucleotide phosphoramidate prodrugs.

The bacterial enzyme phosphotriesterase (PTE) is noted for its ability to hydrolyze many organophosphate compounds, including insecticides and chemical warfare agents. PTE has been the subject of multiple enzyme evolution attempts, which have been highly successful against specific insecticides and the G-type nerve agents. Similar attempts targeting the V-type nerve agents have failed to achieve the same degree of success. Enzyme evolution is an inherently complex problem, which is complicated by synergistic effects, the need to use analogues in high-throughput screening, and a lack of quantitative data to direct future efforts. Previous evolution experiments with PTE have assumed an absence of synergy and minimally screened large libraries, which provides no quantitative information about the effects of individual mutations. Here a systemic approach has been applied to a 28800-member six-site PTE library. The library is screened against multiple V-agent analogues, and a combination of sequence and quantitative activity analysis is used to extract data about the effects of individual mutations. We demonstrate that synergistic relationships dominate the evolutionary landscape of PTE and that analogue activity profiles can be used to identify variants with high activity for substrates. Using these approaches, multiple variants with k cat / K m values for the hydrolysis of VX that were improved >1500-fold were identified, including one variant that is improved 9200-fold relative to wild-type PTE and is specific for the S P enantiomer of VX. Multiple variants that were highly active for ( S P )-VR were identified, the best of which has a k cat / K m values that is improved 13400-fold relative to that of wild-type PTE.

Organophosphorus flame retardants are stable toxic compounds used in nearly all durable plastic products and are considered major emerging pollutants. The phosphotriesterase from Sphingobium sp. TCM1 ( Sb-PTE) is one of the few enzymes known to be able to hydrolyze organophosphorus flame retardants such as triphenyl phosphate and tris(2-chloroethyl) phosphate. The effectiveness of Sb-PTE for the hydrolysis of these organophosphates appears to arise from its ability to hydrolyze unactivated alkyl and phenolic esters from the central phosphorus core. How Sb-PTE is able to catalyze the hydrolysis of the unactivated substituents is not known. To interrogate the catalytic hydrolysis mechanism of Sb-PTE, the pH dependence of the reaction and the effects of changing the solvent viscosity were determined. These experiments were complemented by measurement of the primary and secondary 18-oxygen isotope effects on substrate hydrolysis and a determination of the effects of changing the p K a of the leaving group on the magnitude of the rate constants for hydrolysis. Collectively, the results indicated that a single group must be ionized for nucleophilic attack and that a separate general acid is not involved in protonation of the leaving group. The Brønsted analysis and the heavy atom kinetic isotope effects are consistent with an early associative transition state with subsequent proton transfers not being rate limiting. A novel binding mode of the substrate to the binuclear metal center and a catalytic mechanism are proposed to explain the unusual ability of Sb-PTE to hydrolyze unactivated esters from a wide range of organophosphate substrates.

The phosphotriesterase homology protein (PHP) from Escherichia coli is a member of a family of proteins that is related to phosphotriestrase (PTE), a bacterial enzyme from cog1735 with unusual substrate specificity toward the hydrolysis of synthetic organic phosphates and phosphonates. PHP was cloned, purified to homogeneity, and functionally characterized. The three-dimensional structure of PHP was determined at a resolution of 1.84 Å with zinc and phosphate in the active site. The protein folds as a distorted (β/α) 8 -barrel and possesses a binuclear metal center in the active site. The catalytic function and substrate profile of PHP were investigated using a structure-guided approach that combined bioinformatics, computational docking, organic synthesis, and steady-state enzyme kinetics. PHP was found to catalyze the hydrolysis of phosphorylated glyceryl acetates. The best substrate was 1,2-diacetyl glycerol-3-phosphate with a k cat / K m of 4.9 × 10 3 M -1 s -1 . The presence of a phosphate group in the substrate was essential for enzymatic hydrolysis by the enzyme. It was surprising, however, to find that PHP was unable to hydrolyze any of the lactones tested as potential substrates, unlike most of the other enzymes from cog1735.

Potassium ion channels are emerging as promalignant factors involved in cancer progression. In this study, we found that invading human gastric cancer cells express high levels of inwardly rectifying potassium channel 2.1 (Kir2.1). Silencing Kir2.1 markedly reduced the invasive and metastatic capabilities as well as the epithelial-mesenchymal transition (EMT) of gastric cancer cells. The promalignant nature of Kir2.1 in gastric cancer cells was independent of potassium permeation but relied on its interaction with serine/threonine-protein kinase 38 (Stk38) to inhibit ubiquitination and degradation of mitogen-activated protein kinase kinase kinase 2 (MEKK2). Degradation of MEKK2 was mediated by small mothers against decapentaplegic-specific E3 ubiquitin protein ligase 1 (Smurf1), which resulted in activation of the MEK1/2-ERK1/2-Snail pathway in gastric cancer cells. In human gastric cancer tissues, expression was high and positively correlated with invasion depth and metastatic status of the tumors as well as poor overall patient survival. Cox regression analysis identified Kir2.1 as an independent prognostic indicator for patients with gastric cancer. Our results suggest that Kir2.1 is an important regulator of gastric cancer malignancy and acts as a novel prognostic marker and a therapeutic target for gastric cancer. Significance: Kir2.1 contributes to invasion and metastasis by a noncanonical ion permeation-independent signaling pathway and may act as a novel prognostic marker and therapeutic target for gastric cancer. Cancer Res; 78(11); 3041-53. ©2018 AACR ., (©2018 American Association for Cancer Research.)

The phosphotriesterase from Sphingobium sp. TCM1 ( Sb-PTE) is notable for its ability to hydrolyze organophosphates that are not substrates for other enzymes. In an attempt to determine the catalytic properties of Sb-PTE for hydrolysis of chiral phosphotriesters, we discovered that multiple phosphodiester products are formed from a single substrate. For example, Sb-PTE catalyzes the hydrolysis of the R P -enantiomer of methyl cyclohexyl p-nitrophenyl phosphate with exclusive formation of methyl cyclohexyl phosphate. However, the enzyme catalyzes hydrolysis of the S P -enantiomer of this substrate to an equal mixture of methyl cyclohexyl phosphate and cyclohexyl p-nitrophenyl phosphate products. The ability of this enzyme to catalyze the hydrolysis of a methyl ester at the same rate as the hydrolysis of a p-nitrophenyl ester contained within the same substrate is remarkable. The overall scope of the stereoselective properties of this enzyme is addressed with a library of chiral and prochiral substrates.

Formyl peptide receptor 2 (FPR2), a classical chemoattractant receptor of G-protein-coupled receptors, is reported to be involved in invasion and metastasis of some cancers, but the role of FPR2 in gastric cancer (GC) has not yet been elucidated. In this study, we found that the levels of FPR2 expression in GC were positively correlated with invasion depth, lymph node metastasis and negatively correlated with the patients' overall survival. Multivariate analysis indicated that FPR2 expression was an independent prognostic marker for GC patients. FPR2-knockdown significantly abrogated the migration and invasion stimulated by Hp(2-20) and Ac(2-26), two well-characterized ligands for FPR2 in GC cells. FPR2 deletion also reduced the tumorigenic and metastatic capabilities of GC cells in vivo. Mechanistically, stimulation with FPR2 ligands resulted in down-regulation of E-cadherin and up-regulation of vimentin, which were reversed by FPR2 knock-down, implying the involvement of epithelial-mesenchymal transition (EMT). Moreover, the activation of FPR2 was accompanied with ERK1/2 phosphorylation, which could be attenuated by FPR2 silencing or treatment with MEK inhibitor, PD98059. Altogether, our results demonstrate that FPR2 is functionally involved in invasion and metastasis, and potentially acts as a novel prognostic marker as well as a potential therapeutic target in human GC.

A novel phosphotriesterase was recently discovered and purified from Sphingobium sp. TCM1 (Sb-PTE) and shown to catalyze the hydrolysis of a broad spectrum of organophosphate esters with a catalytic efficiency that exceeds 10(6) M(-1) s(-1) for the hydrolysis of triphenyl phosphate. The enzyme was crystallized and the three-dimensional structure determined to a resolution of 2.1 Å using single-wavelength anomalous diffraction (Protein Data Bank entry 5HRM ). The enzyme adopts a seven-bladed β-propeller protein fold, and three disulfide bonds were identified between Cys-146 and Cys-242, Cys-411 and Cys-443, and Cys-542 and Cys-559. The active site of Sb-PTE contains a binuclear manganese center that is nearly identical to that of the structurally unrelated phosphotriesterase from Pseudomonas diminuta (Pd-PTE). The two metal ions in the active site are bridged to one another by Glu-201 and a water molecule. The α-metal ion is further coordinated to the protein by interactions with His-389, His-475, and Glu-407, whereas the β-metal ion is further liganded to His-317 and His-258. Computational docking of mimics of the proposed pentavalent reaction intermediates for the hydrolysis of organophosphates was used to provide a model for the binding of chiral substrates in the active site of Sb-PTE. The most striking difference in the catalytic properties of Sb-PTE, relative to those of Pd-PTE, is the enhanced rate of hydrolysis of organophosphate esters with substantially weaker leaving groups. The structural basis for this difference in the catalytic properties between Sb-PTE and Pd-PTE, despite the nearly identical binuclear metal centers for the activation of the substrate and nucleophilic water molecule, is at present unclear.

Invasion and metastasis are major malignant characteristics of human gastric cancer (GC), but the underlying molecular mechanisms are poorly understood. Recent studies have shown that scinderin (SCIN), an actin severing and capping protein that regulates the actin cytoskeleton, is involved in the proliferation and migration of certain cancer cells. Accordingly, this study aimed to investigate the potential role of SCIN in the invasion and metastasis of human GC cells and to evaluate its prognostic value for GC patients. We found that high levels of SCIN expression in GC tumors were correlated with poor overall survival of patients. Silencing of SCIN effectively suppressed the migratory and invasive capabilities of human GC cells in vitro and tumorigenicity and metastasis in vivo. Furthermore, knockdown of SCIN markedly inhibited the formation of filopodia, decreasing GC cell migration and the expression of Cdc42, an important regulator of filopodia by GC cells. These findings suggest that SCIN may be a novel prognostic marker and a potential therapeutic target in human GC., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Runt-related transcription factor 2 (RUNX2) is a regulator of embryogenesis and development, but has also been implicated in the progression of certain human cancer. This study aimed to elucidate the role of RUNX2 in the invasive and metastatic potentials of human gastric cancer (GC) and the underlying mechanisms. We found that the levels of RUNX2 expression in gastric cancer tissues were correlated with the differentiation degrees, invasion depth and lymph node metastasis. COX regression analysis indicated that RUNX2 was an independent prognostic indicator for GC patients. RUNX2 significantly increased the migration and invasion ability of GC cells in vitro and enhanced the invasion and metastatic potential of GC cells in an orthotopic GC model of nude mice. Mechanistically, RUNX2 directly bound to the promoter region of the gene coding for the chemokine receptor CXCR4 to enhance its transcription. CXCR4 knockdown or treatment with AMD3100, a CXCR4 inhibitor, attenuated RUNX2-promoted invasion and metastasis. These results demonstrate that RUNX2 promotes the invasion and metastasis of human GC by transcriptionally up-regulating the chemokine receptor CXCR4. Therefore, the RUNX2-CXCR4 axis is a potential therapeutic target for GC.

The mechanism of action of the manganese-dependent phosphotriesterase from Sphingobium sp. strain TCM1 that is capable of hydrolyzing organophosphate flame retardants was determined. The enzyme was shown to hydrolyze the RP-enantiomer of O-methyl O-cyclohexyl p-nitrophenyl thiophosphate with net inversion of configuration and without the formation of a covalent reaction intermediate. These results demonstrate that the enzyme catalyzes the hydrolysis of substrates by activation of a nucleophilic water molecule for direct attack at the phosphorus center.

The most familiar organophosphorus compounds are the neurotoxic insecticides and nerve agents. A related group of organophosphorus compounds, the phosphotriester plasticizers and flame retardants, has recently become widely used. Unlike the neurotoxic phosphotriesters, the plasticizers and flame retardants lack an easily hydrolyzable bond. While the hydrolysis of the neurotoxic organophosphates by phosphotriesterase enzymes is well-known, the lack of a labile bond in the flame retardants and plasticizers renders them inert to typical phosphotriesterases. A phosphotriesterase from Sphingobium sp. strain TCM1 (Sb-PTE) has recently been reported to catalyze the hydrolysis of organophosphorus flame retardants. This enzyme has now been expressed in Escherichia coli, and the activity with a wide variety of organophosphorus substrates has been characterized and compared to the activity of the well-known phosphotriesterase from Pseudomonas diminuta (Pd-PTE). Structure prediction suggests that Sb-PTE has a β-propeller fold, and homology modeling has identified a potential mononuclear manganese binding site. Sb-PTE exhibits catalytic activity against typical phosphotriesterase substrates such as paraoxon, but unlike Pd-PTE, Sb-PTE is also able to effectively hydrolyze flame retardants, plasticizers, and industrial solvents. Sb-PTE can hydrolyze both phosphorus-oxygen bonds and phosphorus-sulfur bonds, but not phosphorus-nitrogen bonds. The best substrate for Sb-PTE is the flame retardant triphenyl phosphate with a kcat/Km of 1.7 × 10(6) M(-1) s(-1). Quite remarkably, Sb-PTE is also able to hydrolyze phosphotriesters with simple alcohol leaving groups such as tributyl phosphate (kcat/Km = 40 M(-1) s(-1)), suggesting that this enzyme could be useful for the bioremediation of a wide variety of organophosphorus compounds.

Pmi1525, an enzyme of unknown function from Proteus mirabilis HI4320 and the amidohydrolase superfamily, was cloned, purified to homogeneity, and functionally characterized. The three-dimensional structure of Pmi1525 was determined with zinc and cacodylate bound in the active site (PDB id: 3RHG ). The structure was also determined with manganese and butyrate in the active site (PDB id: 4QSF ). Pmi1525 folds as a distorted (β/α)8-barrel that is typical for members of the amidohydrolase superfamily and cog1735. The substrate profile for Pmi1525 was determined via a strategy that marshaled the utilization of bioinformatics, structural characterization, and focused library screening. The protein was found to efficiently catalyze the hydrolysis of organophosphonate and carboxylate esters. The best substrates identified for Pmi1525 are ethyl 4-nitrophenylmethyl phosphonate (kcat and kcat/Km values of 580 s(-1) and 1.2 × 10(5) M(-1) s(-1), respectively) and 4-nitrophenyl butyrate (kcat and kcat/Km values of 140 s(-1) and 1.4 × 10(5) M(-1) s(-1), respectively). Pmi1525 is stereoselective for the hydrolysis of chiral methylphosphonate esters. The enzyme hydrolyzes the (SP)-enantiomer of isobutyl 4-nitrophenyl methylphosphonate 14 times faster than the corresponding (RP)-enantiomer. The catalytic properties of this enzyme make it an attractive template for the evolution of novel enzymes for the detection, destruction, and detoxification of organophosphonate nerve agents.

A novel lactonase from Mycoplasma synoviae 53 (MS53_0025) and Mycoplasma agalactiae PG2 (MAG_6390) was characterized by protein structure determination, molecular docking, gene context analysis, and library screening. The crystal structure of MS53_0025 was determined to a resolution of 2.06 Å. This protein adopts a typical amidohydrolase (β/α)8-fold and contains a binuclear zinc center located at the C-terminal end of the β-barrel. A phosphate molecule was bound in the active site and hydrogen bonds to Lys217, Lys244, Tyr245, Arg275, and Tyr278. Both docking and gene context analysis were used to narrow the theoretical substrate profile of the enzyme, thus directing empirical screening to identify that MS53_0025 and MAG_6390 catalyze the hydrolysis of d-xylono-1,4-lactone-5-phosphate (2) with kcat/Km values of 4.7 × 10(4) and 5.7 × 10(4) M(-1) s(-1) and l-arabino-1,4-lactone-5-phosphate (7) with kcat/Km values of 1.3 × 10(4) and 2.2 × 10(4) M(-1) s(-1), respectively. The identification of the substrate profile of these two phospho-furanose lactonases emerged only when all methods were integrated and therefore provides a blueprint for future substrate identification of highly related amidohydrolase superfamily members.

The uncharacterized protein Rsp3690 from Rhodobacter sphaeroides is a member of the amidohydrolase superfamily of enzymes. In this investigation the gene for Rsp3690 was expressed in Escherichia coli and purified to homogeneity, and the three-dimensional structure was determined to a resolution of 1.8 Å. The protein folds as a distorted (β/α)8-barrel, and the subunits associate as a homotetramer. The active site is localized to the C-terminal end of the β-barrel and is highlighted by the formation of a binuclear metal center with two manganese ions that are bridged by Glu-175 and hydroxide. The remaining ligands to the metal center include His-32, His-34, His-207, His-236, and Asp-302. Rsp3690 was shown to catalyze the hydrolysis of a wide variety of carboxylate esters, in addition to organophosphate and organophosphonate esters. The best carboxylate ester substrates identified for Rsp3690 included 2-naphthyl acetate (kcat/Km = 1.0 × 10(5) M(-1) s(-1)), 2-naphthyl propionate (kcat/Km = 1.5 × 10(5) M(-1) s(-1)), 1-naphthyl acetate (kcat/Km = 7.5 × 10(3) M(-1) s(-1)), 4-methylumbelliferyl acetate (kcat/Km = 2.7 × 10(3) M(-1) s(-1)), 4-nitrophenyl acetate (kcat/Km = 2.3 × 10(5) M(-1) s(-1)), and 4-nitrophenyl butyrate (kcat/Km = 8.8 × 10(5) M(-1) s(-1)). The best organophosphonate ester substrates included ethyl 4-nitrophenyl methylphosphonate (kcat/Km = 3.8 × 10(5) M(-1) s(-1)) and isobutyl 4-nitrophenyl methylphosphonate (kcat/Km = 1.1 × 10(4) M(-1) s(-1)). The (SP)-enantiomer of isobutyl 4-nitrophenyl methylphosphonate was hydrolyzed 10 times faster than the less toxic (RP)-enantiomer. The high inherent catalytic activity of Rsp3690 for the hydrolysis of the toxic enantiomer of methylphosphonate esters make this enzyme an attractive target for directed evolution investigations.

The Piwi genes have an important role in stem cell development, gametogenesis and RNA interference in diverse organisms. So far, most of the studies have focused on the function of Piwis in vertebrates, but their function during spermiogenesis in invertebrates still remains largely unclear. In order to investigate the function of Piwis during spermiogenesis in the crab Portunus trituberculatus, we use RT-PCR and RACE to identify three Piwi complete cDNA sequences from the total RNA of the testis in P. trituberculatus. The deduced amino acid sequences of P. trituberculatus Piwi-1, Piwi-2 and Piwi-3 showed that each contains a well-conserved PAZ domain and PIWI domain. RT-PCR analyzed the tissue expression pattern of P. trituberculatus Piwi-1, Piwi-2 and Piwi-3 in the testis, heart, muscle, hepatopancreas and gill. All of the Piwis are found in germ cells of adult testis in P. trituberculatus by in situ hybridization, suggesting that these genes may play function during spermiogenesis in this species., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Metallothioneins (MTs) possess a unique molecular structure that provides metal-binding and redox capabilities. These capabilities include the maintenance of metal equilibria that protect against heavy metals (especially cadmium) and oxidative damage. Past studies have focused on the function of MTs in vertebrates. However, the functions of MTs during spermiogenesis in invertebrates remain unclear. In order to investigate the function of MTs during spermiogenesis in Portunus trituberculatus, we used RT-PCR and RACE to identify two MT complete cDNA sequences in the total RNA from the P. trituberculatus testis. The 450 bp MT-1 cDNA consists of a 77 bp 5' untranslated region, a 196 bp 3' untranslated region, and a 177 bp open reading frame that encodes 58 amino acids including 19 cysteines. The 581 bp MT-2 cDNA consists of 73 bp 5' untranslated region, a 328 bp 3' untranslated region, and a 180 bp open reading frame that encodes 59 amino acids including 18 cysteines. MT-1 and MT-2 of P. trituberculatus more closely resemble invertebrate (especially crab) MT homologues than vertebrate MT homologues as indicated by protein alignment comparisons and phylogenetic tree analysis. MT-1 and MT-2 were detected in the heart, testis, muscle, hepatopancreas, and gill of P. trituberculatus by tissue expression analysis. In addition, MT-1 and MT-2 are present during the entire process of spermiogenesis in P. trituberculatus as indicated by H&E staining and in situ hybridization. MT-1 and MT-2 expression levels significantly increase after cadmium (Cd) exposure as measured by real-time quantitative PCR analysis. Therefore, we suggest that MT-1 and MT-2 perform important functions in spermiogenesis and testis detoxification in P. trituberculatus., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Two enzymes of unknown function from the cog1735 subset of the amidohydrolase superfamily (AHS), LMOf2365_2620 (Lmo2620) from Listeria monocytogenes str. 4b F2365 and Bh0225 from Bacillus halodurans C-125, were cloned, expressed, and purified to homogeneity. The catalytic functions of these two enzymes were interrogated by an integrated strategy encompassing bioinformatics, computational docking to three-dimensional crystal structures, and library screening. The three-dimensional structure of Lmo2620 was determined at a resolution of 1.6 Å with two phosphates and a binuclear zinc center in the active site. The proximal phosphate bridges the binuclear metal center and is 7.1 Å from the distal phosphate. The distal phosphate hydrogen bonds with Lys-242, Lys-244, Arg-275, and Tyr-278. Enzymes within cog1735 of the AHS have previously been shown to catalyze the hydrolysis of substituted lactones. Computational docking of the high-energy intermediate form of the KEGG database to the three-dimensional structure of Lmo2620 highly enriched anionic lactones versus other candidate substrates. The active site structure and the computational docking results suggested that probable substrates would likely include phosphorylated sugar lactones. A small library of diacid sugar lactones and phosphorylated sugar lactones was synthesized and tested for substrate activity with Lmo2620 and Bh0225. Two substrates were identified for these enzymes, D-lyxono-1,4-lactone-5-phosphate and l-ribono-1,4-lactone-5-phosphate. The k(cat)/K(m) values for the cobalt-substituted enzymes with these substrates are ~10(5) M(-1) s(-1).

Objective: To compare the effect of abdominal acupuncture and Chinese medicine on pain relieving in pelvic cavity in patients with endometriosis., Methods: Fifty-eight cases were randomly divided into 2 groups. Thirty cases were in abdominal acupuncture group and 28 cases in Chinese medicine group. Abdominal acupuncture points such as Zhongwan (CV 12), Xiawan (CV 10) and Qihai (CV 6), etc. were adopted for the abdominal acupuncture group, and Tianqi Tongjing Capsule (radix notoginseng capsule for dysmenorrhea) was taken by the Chinese medicine group. After a 3-month treatment, the scores of McGill pain questionaire, level of serum CA125, average value of the radial line of endometrial cyst of ovary and the sum of 3 radial lines of the uterus of patients with adenomyosis as the complication of both groups were observed before and after treatment., Results: The McGill estimation of 6 items for both groups improved obviously after treatment (all P < 0.01, except numbers of selected deseriptors in Chinese medicine group). The differences of the result of McGill estimation of 6 items after treatment had statistical significance, the scores in the abdominal acupuncture group were obviously better than those in the Chinese medicine group (all P < 0.01). The differences of CA125 levels within one group or between 2 groups had statistical significance (P < 0.01, P < 0.05). The difference of the radial lines of patients with endometrial cyst of ovary within one group or between 2 groups after treatment had not statistical significance (all P > 0.05). For the value of 3 radial lines of the uterus of patients with adenomyosis within one group before and after treatment, only the difference in the abdominal acupuncture group had statistical significance (P < 0.01). The differences before and after treatment in the Chinese medicine group and the difference between 2 groups after treatment had no statistical significance (all P > 0.05)., Conclusion: Effect of abdominal acupuncture on relieving pain of pelvic cavity caused by endometriosis, reducing the level of serum CA125 is obverious than Tianqi Tongjing Capsule (radix notoginseng capsule for dysmenorrhea). However, the effects on reducing the size of the ovarian endometrial cyst and the size of uterus with adenomyosis are not significant. Therefore, it is concluded that abdominal acupuncture is a better choice for endometriosis with pain as the chief complaints.

Two uncharacterized enzymes from the amidohydrolase superfamily belonging to cog1228 were cloned, expressed, and purified to homogeneity. The two proteins, Sgx9260c ( gi|44242006 ) and Sgx9260b ( gi|44479596 ), were derived from environmental DNA samples originating from the Sargasso Sea. The catalytic function and substrate profiles for Sgx9260c and Sgx9260b were determined using a comprehensive library of dipeptides and N-acyl derivative of l-amino acids. Sgx9260c catalyzes the hydrolysis of Gly-l-Pro, l-Ala-l-Pro, and N-acyl derivatives of l-Pro. The best substrate identified to date is N-acetyl-l-Pro with a value of k(cat)/K(m) of 3 x 10(5) M(-1) s(-1). Sgx9260b catalyzes the hydrolysis of l-hydrophobic l-Pro dipeptides and N-acyl derivatives of l-Pro. The best substrate identified to date is N-propionyl-l-Pro with a value of k(cat)/K(m) of 1 x 10(5) M(-1) s(-1). Three-dimensional structures of both proteins were determined by X-ray diffraction methods (PDB codes 3MKV and 3FEQ ). These proteins fold as distorted (beta/alpha)(8)-barrels with two divalent cations in the active site. The structure of Sgx9260c was also determined as a complex with the N-methylphosphonate derivative of l-Pro (PDB code 3N2C ). In this structure the phosphonate moiety bridges the binuclear metal center, and one oxygen atom interacts with His-140. The alpha-carboxylate of the inhibitor interacts with Tyr-231. The proline side chain occupies a small substrate binding cavity formed by residues contributed from the loop that follows beta-strand 7 within the (beta/alpha)(8)-barrel. A total of 38 other proteins from cog1228 are predicted to have the same substrate profile based on conservation of the substrate binding residues. The structure of an evolutionarily related protein, Cc2672 from Caulobacter crecentus, was determined as a complex with the N-methylphosphonate derivative of l-arginine (PDB code 3MTW ).

Two proteins from the amidohydrolase superfamily of enzymes were cloned, expressed, and purified to homogeneity. The first protein, Cc0300, was from Caulobacter crescentus CB-15 (Cc0300), while the second one (Sgx9355e) was derived from an environmental DNA sequence originally isolated from the Sargasso Sea ( gi|44371129 ). The catalytic functions and the substrate profiles for the two enzymes were determined with the aid of combinatorial dipeptide libraries. Both enzymes were shown to catalyze the hydrolysis of l-Xaa-l-Xaa dipeptides in which the amino acid at the N-terminus was relatively unimportant. These enzymes were specific for hydrophobic amino acids at the C-terminus. With Cc0300, substrates terminating in isoleucine, leucine, phenylalanine, tyrosine, valine, methionine, and tryptophan were hydrolyzed. The same specificity was observed with Sgx9355e, but this protein was also able to hydrolyze peptides terminating in threonine. Both enzymes were able to hydrolyze N-acetyl and N-formyl derivatives of the hydrophobic amino acids and tripeptides. The best substrates identified for Cc0300 were l-Ala-l-Leu with k(cat) and k(cat)/K(m) values of 37 s(-1) and 1.1 x 10(5) M(-1) s(-1), respectively, and N-formyl-l-Tyr with k(cat) and k(cat)/K(m) values of 33 s(-1) and 3.9 x 10(5) M(-1) s(-1), respectively. The best substrate identified for Sgx9355e was l-Ala-l-Phe with k(cat) and k(cat)/K(m) values of 0.41 s(-1) and 5.8 x 10(3) M(-1) s(-1). The three-dimensional structure of Sgx9355e was determined to a resolution of 2.33 A with l-methionine bound in the active site. The alpha-carboxylate of the methionine is ion-paired to His-237 and also hydrogen bonded to the backbone amide groups of Val-201 and Leu-202. The alpha-amino group of the bound methionine interacts with Asp-328. The structural determinants for substrate recognition were identified and compared with other enzymes in this superfamily that hydrolyze dipeptides with different specificities.

The substrate profiles for two proteins from Caulobacter crescentus CB15 (Cc2672 and Cc3125) and one protein (Sgx9359b) derived from a DNA sequence ( gi|44368820 ) isolated from the Sargasso Sea were determined using combinatorial libraries of dipeptides and N-acyl derivatives of amino acids. These proteins are members of the amidohydrolase superfamily and are currently misannotated in NCBI as catalyzing the hydrolysis of l-Xaa-l-Pro dipeptides. Cc2672 was shown to catalyze the hydrolysis of l-Xaa-l-Arg/Lys dipeptides and the N-acetyl and N-formyl derivatives of lysine and arginine. This enzyme will also hydrolyze longer peptides that terminate in either lysine or arginine. The N-methyl phosphonate derivative of l-lysine was a potent competitive inhibitor of Cc2672 with a K(i) value of 120 nM. Cc3125 was shown to catalyze the hydrolysis of l-Xaa-l-Arg/Lys dipeptides but will not hydrolyze tripeptides or the N-formyl and N-acetyl derivatives of lysine or arginine. The substrate profile for Sgx9359b is similar to that of Cc2672 except that compounds with a C-terminal lysine are not recognized as substrates. The X-ray structure of Sgx9359b was determined to a resolution of 2.3 A. The protein folds as a (beta/alpha)(8)-barrel and self-associates to form a homooctamer. The active site is composed of a binuclear metal center similar to that found in phosphotriesterase and dihydroorotase. In one crystal form, arginine was bound adventitiously to the eight active sites within the octamer. The orientation of the arginine in the active site identified the structural determinants for recognition of the alpha-carboxylate and the positively charged side chains of arginine-containing substrates. This information was used to identify 18 other bacterial sequences that possess identical or similar substrate profiles.

Dr0930, a member of the amidohydrolase superfamily in Deinococcus radiodurans, was cloned, expressed, and purified to homogeneity. The enzyme crystallized in the space group P3121, and the structure was determined to a resolution of 2.1 A. The protein folds as a (beta/alpha)7beta-barrel, and a binuclear metal center is found at the C-terminal end of the beta-barrel. The purified protein contains a mixture of zinc and iron and is intensely purple at high concentrations. The purple color was determined to be due to a charge transfer complex between iron in the beta-metal position and Tyr-97. Mutation of Tyr-97 to phenylalanine or complexation of the metal center with manganese abolished the absorbance in the visible region of the spectrum. Computational docking was used to predict potential substrates for this previously unannotated protein. The enzyme was found to catalyze the hydrolysis of delta- and gamma-lactones with an alkyl substitution at the carbon adjacent to the ring oxygen. The best substrate was delta-nonanoic lactone with a kcat/Km of 1.6 x 10(6) M-1 s-1. Dr0930 was also found to catalyze the very slow hydrolysis of paraoxon with values of kcat and kcat/Km of 0.07 min-1 and 0.8 M-1 s-1, respectively. The amino acid sequence identity to the phosphotriesterase (PTE) from Pseudomonas diminuta is 30%. The eight substrate specificity loops were transplanted from PTE to Dr0930, but no phosphotriesterase activity could be detected in the chimeric PTE-Dr0930 hybrid. Mutation of Phe-26 and Cys-72 in Dr0930 to residues found in the active site of PTE enhanced the kinetic constants for the hydrolysis of paraoxon. The F26G/C72I mutant catalyzed the hydrolysis of paraoxon with a kcat of 1.14 min-1, an increase of 16-fold over the wild-type enzyme. These results support previous proposals that phosphotriesterase activity evolved from an ancestral parent enzyme possessing lactonase activity.

NagA is a member of the amidohydrolase superfamily and catalyzes the deacetylation of N-acetyl-d-glucosamine-6-phosphate. The catalytic mechanism of this enzyme was addressed by the characterization of the catalytic properties of metal-substituted derivatives of NagA from Escherichia coli with a variety of substrate analogues. The reaction mechanism is of interest since NagA from bacterial sources is found with either one or two divalent metal ions in the active site. This observation indicates that there has been a divergence in the evolution of NagA and suggests that there are fundamental differences in the mechanistic details for substrate activation and hydrolysis. NagA from E. coli was inactivated by the removal of the zinc bound to the active site and the apoenzyme reactivated upon incubation with 1 equiv of Zn2+, Cd2+, Co2+, Mn2+, Ni2+, or Fe2+. In the proposed catalytic mechanism the reaction is initiated by the polarization of the carbonyl group of the substrate via a direct interaction with the divalent metal ion and His-143. The invariant aspartate (Asp-273) found at the end of beta-strand 8 in all members of the amidohydrolase superfamily abstracts a proton from the metal-bound water molecule (or hydroxide) to promote the hydrolytic attack on the carbonyl group of the substrate. A tetrahedral intermediate is formed and then collapses with cleavage of the C-N bond after proton transfer to the leaving group amine by Asp-273. The lack of a solvent isotope effect by D2O and the absence of any changes to the kinetic constants with increases in solvent viscosity indicate that net product formation is not limited to any significant extent by proton-transfer steps or the release of products. N-Trifluoroacetyl-d-glucosamine-6-phosphate is hydrolyzed by NagA 26-fold faster than the corresponding N-acetyl derivative. This result is consistent with the formation or collapse of the tetrahedral intermediate as the rate limiting step in the catalytic mechanism of NagA.

Members of the mechanistically diverse enolase superfamily catalyze reactions that are initiated by abstraction of the alpha-proton of a carboxylate anion to generate an enolate anion intermediate that is stabilized by coordination to a Mg2+ ion. The catalytic groups, ligands for an essential Mg2+ and acid/base catalysts, are located in the (beta/alpha)8-barrel domain of the bidomain proteins. The assigned physiological functions in the muconate lactonizing enzyme (MLE) subgroup (Lys acid/base catalysts at the ends of the second and sixth beta-strands in the barrel domain) are cycloisomerization (MLE), dehydration (o-succinylbenzoate synthase; OSBS), and epimerization (L-Ala-D/L-Glu epimerase). We previously studied a putatively promiscuous member of the MLE subgroup with uncertain physiological function from Amycolatopsis that was discovered based on its ability to catalyze the racemization of N-acylamino acids (N-acylamino acid racemase; NAAAR) but also catalyzes the OSBS reaction [OSBS/NAAAR; Palmer, D. R., Garrett, J. B., Sharma, V., Meganathan, R., Babbitt, P. C., and Gerlt, J. A. (1999) Biochemistry 38, 4252-4258]. In this manuscript, we report functional characterization of a homologue of this protein encoded by the genome of Geobacillus kaustophilus as well as two other proteins that are encoded by the same operon, a divergent member of the Gcn5-related N-acetyltransferase (GNAT) superfamily of enzymes whose members catalyze the transfer an acyl group from an acyl-CoA donor to an amine acceptor, and a member of the M20 peptidase/carboxypeptidase G2 family. We determined that the member of the GNAT superfamily is succinyl-CoA:D-amino acid N-succinyltransferase, the member of the enolase superfamily is N-succinylamino acid racemase (NSAR), and the member of the M20 peptidase/carboxypeptidase G2 family is N-succinyl-L-amino acid hydrolase. We conclude that (1) these enzymes constitute a novel, irreversible pathway for the conversion of D- to L-amino acids and (2) the NSAR reaction is a new physiological function in the MLE subgroup. The NSAR is also functionally promiscuous and catalyzes an efficient OSBS reaction; intriguingly, the operon for menaquinone biosynthesis in G. kaustophilus does not encode an OSBS, raising the possibility that the NSAR is a bifunctional enzyme rather than an accidentally promiscuous enzyme.

Reactions of the unsymmetrical phenol ligand 2-(bis(2-pyridylmethyl)aminomethyl)-6-((2-pyridylmethyl)(benzyl)aminomethyl)-4-methylphenol with Mn(OAc)(2).4H(2)O or Mn(H(2)O)(6)(ClO(4))(2) in the presence of NaOBz affords the dimanganese(II) complexes 1(CH(3)OH), [Mn(2)(L)(OAc)(2)(CH(3)OH)](ClO(4)), and 2(H(2)O), [Mn(2)(L)(OBz)(2)(H(2)O)](ClO(4)), respectively. On the other hand, reaction of the ligand with hydrated manganese(III) acetate furnishes the mixed-valent derivative 3(H(2)O), [Mn(2)(L)(OAc)(2)(H(2)O)](ClO(4))( 2). The three complexes have been characterized by X-ray crystallography. 1(CH(3)OH) crystallizes in the monoclinic system, space group P2(1)/c, with a = 10.9215(6) A, b = 20.2318(12) A, c = 19.1354(12) A, alpha = 90 degrees, beta = 97.5310(10) degrees, gamma = 90 degrees, V = 4191.7 A(3), and Z = 4. 2(H(2)O) crystallizes in the monoclinic system, space group P2(1)/n, with a = 10.9215(6) A, b = 20.2318(12) A, c = 19.1354(12) A, alpha = 90 degrees, beta = 97.5310(10) degrees, gamma = 90 degrees, V = 4191.7 A(3), and Z = 4. 3(H(2)O) crystallizes in the monoclinic system, space group P2(1)/c, with a = 11.144(6) A, b = 18.737(10) A, c = 23.949(13) A, alpha = 90 degrees, beta = 95.910(10) degrees, gamma = 90 degrees, V = 4974(5) A(3), and Z = 4. Magnetic measurements revealed that the three compounds exhibit very similar magnetic exchange interactions -J = 4.3(3) cm(-)(1). They were used to establish tentative magneto-structural correlations which show that for the dimanganese(II) complexes -J decreases when the Mn-O(phenoxo) distance increases as expected from orbital overlap considerations. For the dimanganese(II,III) complexes, crystallographic results show that the Mn(II)-O(phenoxo) and Mn(III)-O(phenoxo) bond lengths are inversely correlated. An interesting magneto-structural correlation is found between -J and the difference between these bond lengths, delta(Mn)(-)(O) = d(Mn)()II(-)(O) - d(Mn)()III(-)(O): the smaller this difference, the larger -J. Electrochemical studies show that the mixed-valence state is favored in 1-3 by ca. 100 mV with respect to analogous complexes of symmetrical ligands, owing to the asymmetry of the electron density as found in the analogous diiron complexes.