Xiang H

References (15)

Title : Novel MAGL Inhibitors Alleviate LPS-Induced Acute Kidney Injury by Inhibiting NLRP3 Inflammatory Vesicles, Modulating Intestinal Flora, Repairing the Intestinal Barrier, and Interfering with Serum Metabolism - Xiang_2023_Molecules_28_7245
Author(s) : Xiang H , Wang Y , Yang L , Liu M , Sun C , Gu Y , Yao J
Ref : Molecules , 28 : , 2023
Abstract : Acute kidney injury (AKI) is a complication of a wide range of serious illnesses for which there is still no better therapeutic agent. We demonstrated that M-18C has a favorable inhibitory effect on monoacylglycerol lipase (MAGL), and several studies have demonstrated that nerve inflammation could be effectively alleviated by inhibiting MAGL, suggesting that M-18C has good anti-inflammatory activity. In this study, we investigated the effect of M-18C on LPS-induced acute kidney injury (AKI), both in vivo and in vitro, by using liquid chromatography-mass spectrometry (LC-MS), 16S rRNA gene sequencing, Western blot, and immunohistochemistry. The results showed that both in vivo and in vitro M-18C reduced the release of TNF-alpha and IL-1beta by inhibiting the expression of NOD-like receptor thermal protein domain-associated protein 3 (NLRP3) and apoptosis-associated speck-like protein containing a CARD (ASC) protein; in addition, M-18C was able to intervene in LPS-induced AKI by ameliorating renal pathological injury, repairing the intestinal barrier, and regulating gut bacterial flora and serum metabolism. In conclusion, this study suggests that M-18C has the potential to be a new drug for the treatment of AKI.
ESTHER : Xiang_2023_Molecules_28_7245
PubMedSearch : Xiang_2023_Molecules_28_7245
PubMedID: 37959665
Gene_locus related to this paper: human-MGLL

Title : Characterization of Feruloyl Esterase from Klebsiella oxytoca Z28 and Its Application in the Release of Ferulic Acid from De-Starching Wheat Bran - Zhang_2023_Microorganisms_11_989
Author(s) : Zhang Y , Feng Z , Xiang H , Zhang X , Yang L
Ref : Microorganisms , 11 :989 , 2023
Abstract : Feruloyl esterase (EC3.1.1.73; FAE) can degrade biomass to release ferulic acid (FA), which has a high application in bioprocessing, food, pharmaceutical, paper, feed, and other industrial fields. A strain of Klebsiella oxytoca Z28 with ferulic esterase activity was screened from Daqu. In addition, the FAE gene was expressed in Escherichia coli BL21 (DE3). The enzyme consists of 340 amino acids with a molecular mass of 37.7 kDa. The FAE enzyme activity was 463 U/L when the substrate was ethyl 4-hydroxy-3-methoxycinnamate and the optimum temperature and pH were 50 degreesC and 8.0, respectively. The enzyme had good stability at temperatures of 25-40 degreesC and a pH of 8.0. Ba(2+), Cu(2+), Mn(2+), and Ca(2+) had a strong inhibitory effect on the enzyme activity, and Na(+) had a promotive effect on the enzyme activity. The de-starching wheat bran was degraded by KoFAE, and the FA release was up to 227.15 microg/g. This indicated that the heterologous expression of KoFAE from Klebsiella oxytoca Z28 in E. coli had a certain potential of biodegradation, which can be applied to the degradation of agricultural waste to obtain high value-added FA products.
ESTHER : Zhang_2023_Microorganisms_11_989
PubMedSearch : Zhang_2023_Microorganisms_11_989
PubMedID: 37110412
Gene_locus related to this paper: kleox-KoFae

Title : Computational redesign of a PETase for plastic biodegradation by the GRAPE strategy - Cui_2020_Biorxiv__
Author(s) : Cui YL , Chen YC , Liu XY , Dong SJ , Han J , Xiang H , Chen Q , Liu HY , Han X , Liu WD , Tang SY , Wu B
Ref : Biorxiv , : , 2020
Abstract : The excessive use of plastics has been accompanied by severe ecologically damaging effects. The recent discovery of a PETase from Ideonella sakaiensis that decomposes poly(ethylene terephthalate) (PET) under mild conditions provides an attractive avenue for the biodegradation of plastics. However, the inherent instability of the enzyme limits its practical utilization. Here, we devised a novel computational strategy (greedy accumulated strategy for protein engineering, GRAPE). A systematic clustering analysis combined with greedy accumulation of beneficial mutations in a computationally derived library enabled the design of a variant, DuraPETase, which exhibits an apparent melting temperature that is drastically elevated by 31C and strikingly enhanced degradation performance toward semicrystalline PET films (23%) at mild temperatures (over two orders of magnitude improvement). The mechanism underlying the robust promotion of enzyme performance has been demonstrated via a crystal structure and molecular dynamics simulations. This work shows the capabilities of computational enzyme design to circumvent antagonistic epistatic effects and provides a valuable tool for further understanding and advancing polyester hydrolysis in the natural environment
ESTHER : Cui_2020_Biorxiv__
PubMedSearch : Cui_2020_Biorxiv__
PubMedID:
Gene_locus related to this paper: idesa-peth

Title : Outbred genome sequencing and CRISPR\/Cas9 gene editing in butterflies - Li_2015_Nat.Commun_6_8212
Author(s) : Li X , Fan D , Zhang W , Liu G , Zhang L , Zhao L , Fang X , Chen L , Dong Y , Chen Y , Ding Y , Zhao R , Feng M , Zhu Y , Feng Y , Jiang X , Zhu D , Xiang H , Feng X , Li S , Wang J , Zhang G , Kronforst MR , Wang W
Ref : Nat Commun , 6 :8212 , 2015
Abstract : Butterflies are exceptionally diverse but their potential as an experimental system has been limited by the difficulty of deciphering heterozygous genomes and a lack of genetic manipulation technology. Here we use a hybrid assembly approach to construct high-quality reference genomes for Papilio xuthus (contig and scaffold N50: 492 kb, 3.4 Mb) and Papilio machaon (contig and scaffold N50: 81 kb, 1.15 Mb), highly heterozygous species that differ in host plant affiliations, and adult and larval colour patterns. Integrating comparative genomics and analyses of gene expression yields multiple insights into butterfly evolution, including potential roles of specific genes in recent diversification. To functionally test gene function, we develop an efficient (up to 92.5%) CRISPR/Cas9 gene editing method that yields obvious phenotypes with three genes, Abdominal-B, ebony and frizzled. Our results provide valuable genomic and technological resources for butterflies and unlock their potential as a genetic model system.
ESTHER : Li_2015_Nat.Commun_6_8212
PubMedSearch : Li_2015_Nat.Commun_6_8212
PubMedID: 26354079
Gene_locus related to this paper: papxu-a0a194pj15 , papxu-a0a194q254 , papma-a0a194rdx2 , papxu-a0a194q858 , papxu-a0a194pyl3 , papxu-a0a194q337 , papma-a0a194r1p9 , papma-a0a194r6h1 , papxu-a0a194q1w8 , papma-a0a194ql80 , papma-a0a0n1ipl3 , papma-a0a194qm14

Title : Whole-genome sequence of a flatfish provides insights into ZW sex chromosome evolution and adaptation to a benthic lifestyle - Chen_2014_Nat.Genet_46_253
Author(s) : Chen S , Zhang G , Shao C , Huang Q , Liu G , Zhang P , Song W , An N , Chalopin D , Volff JN , Hong Y , Li Q , Sha Z , Zhou H , Xie M , Yu Q , Liu Y , Xiang H , Wang N , Wu K , Yang C , Zhou Q , Liao X , Yang L , Hu Q , Zhang J , Meng L , Jin L , Tian Y , Lian J , Yang J , Miao G , Liu S , Liang Z , Yan F , Li Y , Sun B , Zhang H , Zhu Y , Du M , Zhao Y , Schartl M , Tang Q , Wang J
Ref : Nat Genet , 46 :253 , 2014
Abstract : Genetic sex determination by W and Z chromosomes has developed independently in different groups of organisms. To better understand the evolution of sex chromosomes and the plasticity of sex-determination mechanisms, we sequenced the whole genomes of a male (ZZ) and a female (ZW) half-smooth tongue sole (Cynoglossus semilaevis). In addition to insights into adaptation to a benthic lifestyle, we find that the sex chromosomes of these fish are derived from the same ancestral vertebrate protochromosome as the avian W and Z chromosomes. Notably, the same gene on the Z chromosome, dmrt1, which is the male-determining gene in birds, showed convergent evolution of features that are compatible with a similar function in tongue sole. Comparison of the relatively young tongue sole sex chromosomes with those of mammals and birds identified events that occurred during the early phase of sex-chromosome evolution. Pertinent to the current debate about heterogametic sex-chromosome decay, we find that massive gene loss occurred in the wake of sex-chromosome 'birth'.
ESTHER : Chen_2014_Nat.Genet_46_253
PubMedSearch : Chen_2014_Nat.Genet_46_253
PubMedID: 24487278
Gene_locus related to this paper: cynse-a0a3p8wch2 , cynse-a0a3p8vd14 , cynse-a0a3p8w747 , cynse-a0a3p8wq40 , cynse-a0a3p8wul3 , cynse-a0a3p8vqr4 , cynse-a0a3p8vmz4

Title : Genome sequence of Paenibacillus sp. strain Aloe-11, an endophytic bacterium with broad antimicrobial activity and intestinal colonization ability - Li_2012_J.Bacteriol_194_2117
Author(s) : Li NZ , Xia T , Xu YL , Qiu RR , Xiang H , He D , Peng YY
Ref : Journal of Bacteriology , 194 :2117 , 2012
Abstract : Paenibacillus sp. strain Aloe-11, a Gram-positive, spore-forming, facultatively anaerobic bacterium isolated from the root of Aloe chinensis in the southwest region of China, has excellent antibiotic activity and intestine colonization ability. Here, we present the 5.8-Mb draft genome sequence of Paenibacillus sp. strain Aloe-11.
ESTHER : Li_2012_J.Bacteriol_194_2117
PubMedSearch : Li_2012_J.Bacteriol_194_2117
PubMedID: 22461553
Gene_locus related to this paper: 9bacl-a0a0k2f8v8

Title : Complete genome sequence of the metabolically versatile halophilic archaeon Haloferax mediterranei, a poly(3-hydroxybutyrate-co-3-hydroxyvalerate) producer - Han_2012_J.Bacteriol_194_4463
Author(s) : Han J , Zhang F , Hou J , Liu X , Li M , Liu H , Cai L , Zhang B , Chen Y , Zhou J , Hu S , Xiang H
Ref : Journal of Bacteriology , 194 :4463 , 2012
Abstract : Haloferax mediterranei, an extremely halophilic archaeon, has shown promise for production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) from unrelated cheap carbon sources. Here we report the complete genome (3,904,707 bp) of H. mediterranei CGMCC 1.2087, consisting of one chromosome and three megaplasmids.
ESTHER : Han_2012_J.Bacteriol_194_4463
PubMedSearch : Han_2012_J.Bacteriol_194_4463
PubMedID: 22843593

Title : Complete genome sequence of Haloarcula hispanica, a Model Haloarchaeon for studying genetics, metabolism, and virus-host interaction - Liu_2011_J.Bacteriol_193_6086
Author(s) : Liu H , Wu Z , Li M , Zhang F , Zheng H , Han J , Liu J , Zhou J , Wang S , Xiang H
Ref : Journal of Bacteriology , 193 :6086 , 2011
Abstract : Haloarcula hispanica is an extremely halophilic archaeon that has an unusually low restriction barrier and is therefore significant for studying archaeal genetics, metabolism, and virus-host interactions. Here we report the complete genome sequence (3,890,005 bp) of H. hispanica strain CGMCC 1.2049, consisting of two chromosomes and one megaplasmid.
ESTHER : Liu_2011_J.Bacteriol_193_6086
PubMedSearch : Liu_2011_J.Bacteriol_193_6086
PubMedID: 21994921
Gene_locus related to this paper: halma-q5uym0

Title : Comparison of four phaC genes from Haloferax mediterranei and their function in different PHBV copolymer biosyntheses in Haloarcula hispanica - Han_2010_Saline.Systems_6_9
Author(s) : Han J , Li M , Hou J , Wu L , Zhou J , Xiang H
Ref : Saline Systems , 6 :9 , 2010
Abstract : BACKGROUND: The halophilic archaeon Haloferax mediterranei is able to accumulate large amounts of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with high molar fraction of 3-hydroxyvalerate (3HV) from unrelated carbon sources. A Polyhydroxyalkanoate (PHA) synthase composed of two subunits, PhaCHme and PhaEHme, has been identified in this strain, and shown to account for the PHBV biosynthesis.
RESULTS: With the aid of the genome sequence of Hfx. mediterranei CGMCC 1.2087, three additional phaC genes (designated phaC1, phaC2, and phaC3) were identified, which encoded putative PhaCs. Like PhaCHme (54.8 kDa), PhaC1 (49.7 kDa) and PhaC3 (62.5 kDa) possessed the conserved motifs of type III PHA synthase, which was not observed in PhaC2 (40.4 kDa). Furthermore, the longer C terminus found in the other three PhaCs was also absent in PhaC2. Reverse transcription PCR (RT-PCR) revealed that, among the four genes, only phaCHme was transcribed under PHA-accumulating conditions in the wild-type strain. However, heterologous coexpression of phaEHme with each phaC gene in Haloarcula hispanica PHB-1 showed that all PhaCs, except PhaC2, could lead to PHBV accumulation with various 3HV fractions. The three kinds of copolymers were characterized using gel-permeation chromatography (GPC), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). Their thermal properties changed with the variations in monomer composition as well as the different molecular weights (Mw), thus might meet various application requirements. CONCLUSION: We discover three cryptic phaC genes in Hfx. mediterranei, and demonstrate that genetic engineering of these newly identified phaC genes has biotechnological potential for PHBV production with tailor-made material properties.
ESTHER : Han_2010_Saline.Systems_6_9
PubMedSearch : Han_2010_Saline.Systems_6_9
PubMedID: 20727166

Title : Genetic and biochemical characterization of the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) synthase in Haloferax mediterranei - Lu_2008_J.Bacteriol_190_4173
Author(s) : Lu Q , Han J , Zhou L , Zhou J , Xiang H
Ref : Journal of Bacteriology , 190 :4173 , 2008
Abstract : The haloarchaeon Haloferax mediterranei has shown promise for the economical production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a desirable bioplastic. However, little is known at present about the genes involved in PHBV synthesis in the domain Archaea. In this study, we cloned the gene cluster (phaEC(Hme)) encoding a polyhydroxyalkanoate (PHA) synthase in H. mediterranei CGMCC 1.2087 via thermal asymmetric interlaced PCR. Western blotting revealed that the phaE(Hme) and phaC(Hme) genes were constitutively expressed, and both the PhaE(Hme) and PhaC(Hme) proteins were strongly bound to the PHBV granules. Interestingly, CGMCC 1.2087 could synthesize PHBV in either nutrient-limited medium (supplemented with 1% starch) or nutrient-rich medium, up to 24 or 18% (wt/wt) in shaking flasks. Knockout of the phaEC(Hme) genes in CGMCC 1.2087 led to a complete loss of PHBV synthesis, and only complementation with the phaEC(Hme) genes together (but not either one alone) could restore to this mutant the capability for PHBV accumulation. The known haloarchaeal PhaC subunits are much longer at their C termini than their bacterial counterparts, and the C-terminal extension of PhaC(Hme) was proven to be indispensable for its function in vivo. Moreover, the mixture of purified PhaE(Hme)/PhaC(Hme) (1:1) showed significant activity of PHA synthase in vitro. Taken together, our results indicated that a novel member of the class III PHA synthases, composed of PhaC(Hme) and PhaE(Hme), accounted for the PHBV synthesis in H. mediterranei.
ESTHER : Lu_2008_J.Bacteriol_190_4173
PubMedSearch : Lu_2008_J.Bacteriol_190_4173
PubMedID: 18408025
Gene_locus related to this paper: halme-b3frm5

Title : Novel family of cholesterol esterases produced by actinomycetes bacteria - Xiang_2007_Biochim.Biophys.Acta_1774_112
Author(s) : Xiang H , Masuo S , Hoshino T , Takaya N
Ref : Biochimica & Biophysica Acta , 1774 :112 , 2007
Abstract : Although cholesterol esterase (CHE; EC 3.1.1.13) is widespread in nature, CHEs from Streptomyces lavendulae and Streptomyces sp. X9 are the only known CHEs produced by actinomycetes. We purified CHEs from S. avermitilis JCM5070, and S. griseus IFO13350 and identified four new CHEs from actinomycetes. The enzymic properties of the CHEs from Streptomyces sp. X9, S. avermitilis, and S. griseus including substrate specificity, sensitivity to inhibitors and optimal conditions for catalysis were similar. We identified genes for the CHEs from Streptomyces sp. X9 and S. avermitilis and the encoded predicted sequences comprised 217 and 214 amino acid residues, respectively, with 64% similarity. The CHEs from Streptomyces sp. X9 and S. avermitilis were also 54 and 57% similar, respectively, to S. lavendulae CHE, indicating that these CHEs are orthologs. Phylogenetic analysis showed that they are distantly related to the conventional lipase/esterase type CHEs from mammals, yeasts and other bacteria. The actinomycetes CHEs did not have the Gly-Xaa-Ser-Xaa-Gly sequence that is conserved in the lipase/esterase family. A database search showed that orthologs of this type of CHE were restricted to actinomycetes. These findings imply that the actinomycetes CHEs constitute a novel family of cholesterol esterases.
ESTHER : Xiang_2007_Biochim.Biophys.Acta_1774_112
PubMedSearch : Xiang_2007_Biochim.Biophys.Acta_1774_112
PubMedID: 17161031

Title : Molecular characterization of the phaECHm genes, required for biosynthesis of poly(3-hydroxybutyrate) in the extremely halophilic archaeon Haloarcula marismortui - Han_2007_Appl.Environ.Microbiol_73_6058
Author(s) : Han J , Lu Q , Zhou L , Zhou J , Xiang H
Ref : Applied Environmental Microbiology , 73 :6058 , 2007
Abstract : Although many haloarchaea produce biodegradable polyhydroxyalkanoates (PHAs), the genes involved in PHA synthesis in the domain of Archaea have not yet been experimentally investigated yet. In this study, we revealed that Haloarcula marismortui was able to accumulate poly(3-hydroxybutyrate) (PHB) up to 21% of cellular dry weight when cultured in a minimal medium with excessive glucose and identified the phaE(Hm) and phaC(Hm) genes, probably encoding two subunits of a class III PHA synthase. These two genes were adjacent and directed by a single promoter located 26 bp upstream of the transcriptional start site and were constitutively expressed under both nutrient-rich and -limited conditions. Interestingly, PhaC(Hm) was revealed to be strongly bound with the PHB granules, but PhaE(Hm) seemed not to be. Introduction of either the phaE(Hm) or phaC(Hm) gene into Haloarcula hispanica, which harbors highly homologous phaEC(Hh) genes, could enhance the PHB synthesis in the recombinant strains, while coexpression of the both genes always generated the highest PHB yield. Significantly, knockout of the phaEC(Hh) genes in H. hispanica led to a complete loss of the PHA synthase activity. Complementation with phaEC(Hm) genes, but not a single one, restored the capability of PHB accumulation as well as the PHA synthase activity in this phaEC-deleted haloarchaeon. These results indicated that the phaEC genes are required for biosynthesis of PHB and might encode an active PHA synthase in the Haloarcula species.
ESTHER : Han_2007_Appl.Environ.Microbiol_73_6058
PubMedSearch : Han_2007_Appl.Environ.Microbiol_73_6058
PubMedID: 17675423
Gene_locus related to this paper: halma-q5uym0

Title : Complete genome sequence of the industrial bacterium Bacillus licheniformis and comparisons with closely related Bacillus species - Rey_2004_Genome.Biol_5_R77
Author(s) : Rey MW , Ramaiya P , Nelson BA , Brody-Karpin SD , Zaretsky EJ , Tang M , Lopez de Leon A , Xiang H , Gusti V , Clausen IG , Olsen PB , Rasmussen MD , Andersen JT , Jorgensen PL , Larsen TS , Sorokin A , Bolotin A , Lapidus A , Galleron N , Ehrlich SD , Berka RM
Ref : Genome Biol , 5 :R77 , 2004
Abstract : BACKGROUND: Bacillus licheniformis is a Gram-positive, spore-forming soil bacterium that is used in the biotechnology industry to manufacture enzymes, antibiotics, biochemicals and consumer products. This species is closely related to the well studied model organism Bacillus subtilis, and produces an assortment of extracellular enzymes that may contribute to nutrient cycling in nature.
RESULTS: We determined the complete nucleotide sequence of the B. licheniformis ATCC 14580 genome which comprises a circular chromosome of 4,222,336 base-pairs (bp) containing 4,208 predicted protein-coding genes with an average size of 873 bp, seven rRNA operons, and 72 tRNA genes. The B. licheniformis chromosome contains large regions that are colinear with the genomes of B. subtilis and Bacillus halodurans, and approximately 80% of the predicted B. licheniformis coding sequences have B. subtilis orthologs.
CONCLUSIONS: Despite the unmistakable organizational similarities between the B. licheniformis and B. subtilis genomes, there are notable differences in the numbers and locations of prophages, transposable elements and a number of extracellular enzymes and secondary metabolic pathway operons that distinguish these species. Differences include a region of more than 80 kilobases (kb) that comprises a cluster of polyketide synthase genes and a second operon of 38 kb encoding plipastatin synthase enzymes that are absent in the B. licheniformis genome. The availability of a completed genome sequence for B. licheniformis should facilitate the design and construction of improved industrial strains and allow for comparative genomics and evolutionary studies within this group of Bacillaceae.
ESTHER : Rey_2004_Genome.Biol_5_R77
PubMedSearch : Rey_2004_Genome.Biol_5_R77
PubMedID: 15461803
Gene_locus related to this paper: bacld-q62u01 , bacld-Q65L73 , bacld-q62yz9 , bacld-q65dz7 , bacld-q65e02 , bacld-q65eq1 , bacld-q65fc5 , bacld-q65fg2 , bacld-q65fg3 , bacld-q65fk9 , bacld-q65ft3 , bacld-q65fw3 , bacld-q65fy2 , bacld-q65gx2 , bacld-q65hn8 , bacld-q65hr4 , bacld-q65if8 , bacld-q65iy4 , bacld-q65j72 , bacld-q65le0 , bacld-q65ly2 , bacld-q65m29 , bacld-q65mg8 , bacld-q65my7 , bacld-q65n63 , bacld-q65nk2 , bacld-q65nm7 , bacli-LICC

Title : Thermostable esterase from Thermoanaerobacter tengcongensis: high-level expression, purification and characterization - Zhang_2003_Biotechnol.Lett_25_1463
Author(s) : Zhang J , Liu J , Zhou J , Ren Y , Dai X , Xiang H
Ref : Biotechnol Lett , 25 :1463 , 2003
Abstract : The lipA gene encoding a thermostable esterase was cloned from Thermoanaerobacter tengcongensis and over-expressed in Escherichia coli. The recombinant esterase, with a molecular mass of approx. 43 kDa determined by SDS-PAGE, was purified to homogeneity through Sephadex G-100 gel filtration. The purified enzyme actively hydrolyzed tributyrin but not olive oil. Maximum activity was observed on p-nitrophenyl (NP)-propionate (C3) and p-NP-butyrate (C4), with little activity towards p-NP-palmitate (C16). The esterase was optimally active at 70 degrees C (over 15 min) and at pH 9. It is highly thermostable, with a residual activity greater than 80% after incubation at 50 degrees C for more than 10 h. The activity was not inhibited by 5 mM EDTA and PMSF, indicating the esterase is not a metalloenzyme and may contain a specific structure around the catalytic serine residue. In addition, it was stable for 1 h at 37 degrees C in 1% CHAPS and Triton X-100 but not stable in 1% Tween 20 or SDS.
ESTHER : Zhang_2003_Biotechnol.Lett_25_1463
PubMedSearch : Zhang_2003_Biotechnol.Lett_25_1463
PubMedID: 14514051
Gene_locus related to this paper: thete-LIPA

Title : Identification of active site residues essential to 4-chlorobenzoyl- coenzyme A dehalogenase catalysis by chemical modification and site directed mutagenesis - Yang_1996_Biochemistry_35_10879
Author(s) : Yang G , Liu RQ , Taylor KL , Xiang H , Price J , Dunaway-Mariano D
Ref : Biochemistry , 35 :10879 , 1996
Abstract : 4-Chlorobenzoyl-coenzyme A (4-CBA-CoA) dehalogenase catalyzes the hydrolysis of 4-CBA-CoA to 4-hydroxybenzoyl-coenzyme A (4-HBA-CoA) via a nucleophilic aromatic substitution pathway involving the participation of an active site carboxylate side chain in covalent catalysis. In this paper we report on the identification of conserved aspartate, histidine, and tryptophan residues essential to 4-CBA-CoA catalysis using chemical modification and site-directed mutagenesis techniques. Treatment of the dehalogenase with diethyl pyrocarbonate resulted in complete loss of catalytic activity (Kinact = 0.17 mM-1 min-1 at pH 6.5, 25 degrees C) that was fully regained by subsequent treatment with hydroxylamine. The protection from inactivation afforded by enzyme bound 4-HBA-CoA indicated that the essential histidine residues are located at the active site. Replacement of conserved histidine residues 81, 90, 94, and 208 with glutamine residues resulted in a significant loss of catalytic activity only in the cases of the histidine 81 and 90 mutants. Substrate and product ligand binding studies showed that binding is not significantly inhibited in these mutants. Site directed mutagenesis of a selection of conserved aspartate and glutamate residues, identified aspartate 145 as being essential to dehalogenase catalysis. Ligand binding studies showed that this residue is not required for tight substrate/product binding. Chemical modification of the dehalogenase with N-bromosuccinimide resulted in full loss of catalytic activity that was prevented by saturation of the active site with product ligand, providing evidence favoring an essential active site tryptophan. Phenylalanine replacement of conserved tryptophan residues 179 and 137 reduced catalytic activity only in the latter (Kcat = 0.03% of wild-type dehalogenase). On the basis of these results and the recently determined X-ray crystal structure of the complex of 4-CBA-CoA dehalogenase and 4-HBA-CoA [Benning, M. M., Taylor, K.L., Liu, R.-Q., Yang, G., Xiang, H., Wesenberg, G., Dunaway-Mariano, D., Holden, H.M. (1996) Biochemistry 35,8103-8109] we propose that aspartate 145 functions as the active site nucleophile, that tryptophan 137 serves as a hydrogen bond donor to the aspartate 145 C = O, and that histidine 90 serves to deprotonate the bound H2O molecule.
ESTHER : Yang_1996_Biochemistry_35_10879
PubMedSearch : Yang_1996_Biochemistry_35_10879
PubMedID: 8718880