Brown G

References (10)

Title : Structural insights into hydrolytic defluorination of difluoroacetate by microbial fluoroacetate dehalogenases - Khusnutdinova_2023_FEBS.J_290_4966
Author(s) : Khusnutdinova AN , Batyrova KA , Brown G , Fedorchuk T , Chai YS , Skarina T , Flick R , Petit AP , Savchenko A , Stogios P , Yakunin AF
Ref : Febs J , 290 :4966 , 2023
Abstract : Fluorine forms the strongest single bond to carbon with the highest bond dissociation energy among natural products. However, fluoroacetate dehalogenases (FADs) have been shown to hydrolyze this bond in fluoroacetate under mild reaction conditions. Furthermore, two recent studies demonstrated that the FAD RPA1163 from Rhodopseudomonas palustris can also accept bulkier substrates. In this study, we explored the substrate promiscuity of microbial FADs and their ability to defluorinate polyfluorinated organic acids. Enzymatic screening of eight purified dehalogenases with reported fluoroacetate defluorination activity revealed significant hydrolytic activity against difluoroacetate in three proteins. Product analysis using liquid chromatography-mass spectrometry identified glyoxylic acid as the final product of enzymatic DFA defluorination. The crystal structures of DAR3835 from Dechloromonas aromatica and NOS0089 from Nostoc sp. were determined in the apo-state along with the DAR3835 H274N glycolyl intermediate. Structure-based site-directed mutagenesis of DAR3835 demonstrated a key role for the catalytic triad and other active site residues in the defluorination of both fluoroacetate and difluoroacetate. Computational analysis of the dimer structures of DAR3835, NOS0089, and RPA1163 indicated the presence of one substrate access tunnel in each protomer. Moreover, protein-ligand docking simulations suggested similar catalytic mechanisms for the defluorination of both fluoroacetate and difluoroacetate, with difluoroacetate being defluorinated via two consecutive defluorination reactions producing glyoxylate as the final product. Thus, our findings provide molecular insights into substrate promiscuity and catalytic mechanism of FADs, which are promising biocatalysts for applications in synthetic chemistry and bioremediation of fluorochemicals.
ESTHER : Khusnutdinova_2023_FEBS.J_290_4966
PubMedSearch : Khusnutdinova_2023_FEBS.J_290_4966
PubMedID: 37437000
Gene_locus related to this paper: anasp-ALR0039 , decar-q479b8

Title : Screening and Characterization of Novel Polyesterases from Environmental Metagenomes with High Hydrolytic Activity against Synthetic Polyesters - Hajighasemi_2018_Environ.Sci.Technol_52_12388
Author(s) : Hajighasemi M , Tchigvintsev A , Nocek B , Flick R , Popovic A , Hai T , Khusnutdinova AN , Brown G , Xu X , Cui H , Anstett J , Chernikova TN , Bruls T , Le Paslier D , Yakimov MM , Joachimiak A , Golyshina OV , Savchenko A , Golyshin PN , Edwards EA , Yakunin AF
Ref : Environ Sci Technol , 52 :12388 , 2018
Abstract : The continuous growth of global plastics production, including polyesters, has resulted in increasing plastic pollution and subsequent negative environmental impacts. Therefore, enzyme-catalyzed depolymerization of synthetic polyesters as a plastics recycling approach has become a focus of research. In this study, we screened over 200 purified uncharacterized hydrolases from environmental metagenomes and sequenced microbial genomes and identified at least 10 proteins with high hydrolytic activity against synthetic polyesters. These include the metagenomic esterases MGS0156 and GEN0105, which hydrolyzed polylactic acid (PLA), polycaprolactone, as well as bis(benzoyloxyethyl)-terephthalate. With solid PLA as a substrate, both enzymes produced a mixture of lactic acid monomers, dimers, and higher oligomers as products. The crystal structure of MGS0156 was determined at 1.95 A resolution and revealed a modified alpha/beta hydrolase fold, with a lid domain and highly hydrophobic active site. Mutational studies of MGS0156 identified the residues critical for hydrolytic activity against both polyester and monoester substrates, with two-times higher polyesterase activity in the MGS0156 L169A mutant protein. Thus, our work identified novel, highly active polyesterases in environmental metagenomes and provided molecular insights into their activity, thereby augmenting our understanding of enzymatic polyester hydrolysis.
ESTHER : Hajighasemi_2018_Environ.Sci.Technol_52_12388
PubMedSearch : Hajighasemi_2018_Environ.Sci.Technol_52_12388
PubMedID: 30284819
Gene_locus related to this paper: 9zzzz-a0a0g3fj39 , 9zzzz-a0a0g3fj48 , 9zzzz-A0A0G3FEJ8 , 9bact-a4uz10

Title : Determinants and prediction of esterase substrate promiscuity patterns - Martinez-Martinez_2018_ACS.Chem.Biol_13_225
Author(s) : Martinez-Martinez M , Coscolin C , Santiago G , Chow J , Stogios PJ , Bargiela R , Gertler C , Navarro-Fernandez J , Bollinger A , Thies S , Mendez-Garcia C , Popovic A , Brown G , Chernikova TN , Garcia-Moyano A , Bjergah GE , Perez-Garcia P , Hai T , Del Pozo MV , Stokke R , Steen IH , Cui H , Xu X , Nocek BP , Alcaide M , Distaso M , Mesa V , Pelaez AI , Sanchez J , Buchholz PCF , Pleiss J , Fernandez-Guerra A , Glockner FO , Golyshina OV , Yakimov MM , Savchenko A , Jaeger KE , Yakunin AF , Streit WR , Golyshin PN , Guallar V , Ferrer M
Ref : ACS Chemical Biology , 13 :225 , 2018
Abstract : Esterases receive special attention because their wide distribution in biological systems and environments and their importance for physiology and chemical synthesis. The prediction of esterases substrate promiscuity level from sequence data and the molecular reasons why certain such enzymes are more promiscuous than others, remain to be elucidated. This limits the surveillance of the sequence space for esterases potentially leading to new versatile biocatalysts and new insights into their role in cellular function. Here we performed an extensive analysis of the substrate spectra of 145 phylogenetically and environmentally diverse microbial esterases, when tested with 96 diverse esters. We determined the primary factors shaping their substrate range by analyzing substrate range patterns in combination with structural analysis and protein-ligand simulations. We found a structural parameter that helps ranking (classifying) promiscuity level of esterases from sequence data at 94% accuracy. This parameter, the active site effective volume, exemplifies the topology of the catalytic environment by measuring the active site cavity volume corrected by the relative solvent accessible surface area (SASA) of the catalytic triad. Sequences encoding esterases with active site effective volumes (cavity volume/SASA) above a threshold show greater substrate spectra, which can be further extended in combination with phylogenetic data. This measure provides also a valuable tool for interrogating substrates capable of being converted. This measure, found to be transferred to phosphatases of the haloalkanoic acid dehalogenase superfamily and possibly other enzymatic systems, represents a powerful tool for low-cost bioprospecting for esterases with broad substrate ranges, in large scale sequence datasets.
ESTHER : Martinez-Martinez_2018_ACS.Chem.Biol_13_225
PubMedSearch : Martinez-Martinez_2018_ACS.Chem.Biol_13_225
PubMedID: 29182315
Gene_locus related to this paper: 9zzzz-a0a2k8jn75 , 9zzzz-a0a2k8jt94 , 9zzzz-a0a0g3fj44 , 9zzzz-a0a0g3fh10 , 9zzzz-a0a0g3fh03 , 9bact-a0a1s5qkj8 , 9zzzz-a0a0g3feh5 , 9zzzz-a0a0g3fkz4 , 9zzzz-a0a0g3fh07 , 9zzzz-a0a0g3fh34 , 9zzzz-a0a0g3fh31 , 9bact-KY458167 , alcbs-q0vqa3 , 9bact-a0a1s5qki8 , 9zzzz-a0a0g3feq8 , 9zzzz-a0a0g3feh8 , 9zzzz-a0a0g3fh19 , 9bact-KY203037 , 9bact-a0a1s5ql22 , 9bact-a0a1s5qm34 , 9bact-KY203034 , 9bact-r9qzg0 , 9bact-a0a1s5qly8 , 9zzzz-a0a0g3fkz8 , 9zzzz-a0a0g3feg9 , 9zzzz-KY203033 , 9zzzz-a0a0g3fes4 , 9zzzz-a0a0g3fh42 , 9bact-a0a1s5qlx2 , 9zzzz-KY483651 , 9bact-a0a1s5qmh4 , 9zzzz-KY203032 , 9zzzz-EH87 , 9zzzz-a0a0g3fei1 , 9zzzz-a0a0g3fet2 , 9zzzz-KY483647 , 9zzzz-EH82 , 9zzzz-a0a0g3fe15 , 9bact-KY203031 , 9bact-t1w006 , 9zzzz-a0a0g3fet6 , 9bact-KY458164 , geoth-g8myf3 , 9bact-a0a1s5ql04 , 9gamm-a0a1y0ihk7 , 9bact-a0a1s5qly6 , 9bact-a0a1s5qkg4 , 9bact-a0a1s5qkm4 , 9gamm-s5tv80 , 9gamm-a0a0c4zhg2 , 9zzzz-t1b379 , 9gamm-KY483646 , 9bact-KY458160 , 9zzzz-a0a0g3fj57 , 9gamm-s5t8349 , 9arch-KY203036 , 9bact-KY458168 , 9zzzz-a0a0g3fes0 , 9zzzz-t1be47 , 9bact-KY458159 , 9zzzz-a0a0g3fh39 , 9bact-t1vzd5 , 9prot-EH41 , 9bact-Lip114 , alcbs-q0vt77 , 9bact-a0a1s5qke6 , 9bact-a0a1s5qkf3 , 9prot-SRP030024 , 9gamm-s5t532 , 9bact-a0a1s5qkl2 , 9bact-a0a1s5qkk8 , 9zzzz-KY203030 , 9zzzz-t1d4I7 , 9prot-KY019260 , 9bact-a0a1s5qm38 , 9arch-KY458161 , 9prot-KY010302 , 9zzzz-a0a0g3fl25 , 9actn-KY010298 , 9gamm-s5u059 , 9bact-a0a1s5qmi7 , 9bact-KY010297 , 9bact-KY483642 , 9bact-a0a1s5qkj1 , 9bact-KY010299 , 9bact-KY483648 , alcbs-q0vtl7 , 9bact-a0a1s5qf1 , 9bact-a0a1s5qkg0 , 9bact-a0a0h4tgu6 , 9bact-MilE3 , 9bact-LAE6 , 9alte-MGS-MT1 , 9bact-r9qzf7 , 9gamm-k0c6t6 , alcbs-q0vl36 , alcbs-q0vlq1 , alcbs-q0vq49 , bacsu-pnbae , canar-LipB , canan-lipasA , geost-lipas , marav-a1u5n0 , pseps-i7k8x5 , staep-GEHD , symth-q67mr3 , altma-s5cfn7 , cycsp-k0c2b8 , alcbs-q0vlk5 , 9bact-k7qe48 , 9bact-MGS-M1 , 9bact-MGS-M2 , 9bact-a0a0b5kns5 , 9zzzz-a0a0g3fej4 , 9zzzz-a0a0g3fj60 , 9zzzz-a0a0g3fej0 , 9zzzz-a0a0g3fj64 , 9bact-a0a0b5kc16 , 9zzzz-a0a0g3feg6 , 9zzzz-a0a0g3feu6

Title : Activity screening of environmental metagenomic libraries reveals novel carboxylesterase families - Popovic_2017_Sci.Rep_7_44103
Author(s) : Popovic A , Hai T , Tchigvintsev A , Hajighasemi M , Nocek B , Khusnutdinova AN , Brown G , Glinos J , Flick R , Skarina T , Chernikova TN , Yim V , Bruls T , Paslier DL , Yakimov MM , Joachimiak A , Ferrer M , Golyshina OV , Savchenko A , Golyshin PN , Yakunin AF
Ref : Sci Rep , 7 :44103 , 2017
Abstract : Metagenomics has made accessible an enormous reserve of global biochemical diversity. To tap into this vast resource of novel enzymes, we have screened over one million clones from metagenome DNA libraries derived from sixteen different environments for carboxylesterase activity and identified 714 positive hits. We have validated the esterase activity of 80 selected genes, which belong to 17 different protein families including unknown and cyclase-like proteins. Three metagenomic enzymes exhibited lipase activity, and seven proteins showed polyester depolymerization activity against polylactic acid and polycaprolactone. Detailed biochemical characterization of four new enzymes revealed their substrate preference, whereas their catalytic residues were identified using site-directed mutagenesis. The crystal structure of the metal-ion dependent esterase MGS0169 from the amidohydrolase superfamily revealed a novel active site with a bound unknown ligand. Thus, activity-centered metagenomics has revealed diverse enzymes and novel families of microbial carboxylesterases, whose activity could not have been predicted using bioinformatics tools.
ESTHER : Popovic_2017_Sci.Rep_7_44103
PubMedSearch : Popovic_2017_Sci.Rep_7_44103
PubMedID: 28272521
Gene_locus related to this paper: 9zzzz-a0a0g3fj39 , 9zzzz-a0a0g3fj48 , 9zzzz-A0A0G3FEJ8

Title : Biochemical and Structural Insights into Enzymatic Depolymerization of Polylactic Acid and Other Polyesters by Microbial Carboxylesterases - Hajighasemi_2016_Biomacromolecules_17_2027
Author(s) : Hajighasemi M , Nocek BP , Tchigvintsev A , Brown G , Flick R , Xu X , Cui H , Hai T , Joachimiak A , Golyshin PN , Savchenko A , Edwards EA , Yakunin AF
Ref : Biomacromolecules , 17 :2027 , 2016
Abstract : Polylactic acid (PLA) is a biodegradable polyester derived from renewable resources, which is a leading candidate for the replacement of traditional petroleum-based polymers. Since the global production of PLA is quickly growing, there is an urgent need for the development of efficient recycling technologies, which will produce lactic acid instead of CO2 as the final product. After screening 90 purified microbial alpha/beta-hydrolases, we identified hydrolytic activity against emulsified PLA in two uncharacterized proteins, ABO2449 from Alcanivorax borkumensis and RPA1511 from Rhodopseudomonas palustris. Both enzymes were also active against emulsified polycaprolactone and other polyesters as well as against soluble alpha-naphthyl and p-nitrophenyl monoesters. In addition, both ABO2449 and RPA1511 catalyzed complete or extensive hydrolysis of solid PLA with the production of lactic acid monomers, dimers, and larger oligomers as products. The crystal structure of RPA1511 was determined at 2.2 A resolution and revealed a classical alpha/beta-hydrolase fold with a wide-open active site containing a molecule of polyethylene glycol bound near the catalytic triad Ser114-His270-Asp242. Site-directed mutagenesis of both proteins demonstrated that the catalytic triad residues are important for the hydrolysis of both monoester and polyester substrates. We also identified several residues in RPA1511 (Gln172, Leu212, Met215, Trp218, and Leu220) and ABO2449 (Phe38 and Leu152), which were not essential for activity against soluble monoesters but were found to be critical for the hydrolysis of PLA. Our results indicate that microbial carboxyl esterases can efficiently hydrolyze various polyesters making them attractive biocatalysts for plastics depolymerization and recycling.
ESTHER : Hajighasemi_2016_Biomacromolecules_17_2027
PubMedSearch : Hajighasemi_2016_Biomacromolecules_17_2027
PubMedID: 27087107
Gene_locus related to this paper: marav-a1u5n0 , rhopa-q6n9m9 , alcbs-q0vlq1

Title : The environment shapes microbial enzymes: five cold-active and salt-resistant carboxylesterases from marine metagenomes - Tchigvintsev_2015_Appl.Microbiol.Biotechnol_99_2165
Author(s) : Tchigvintsev A , Tran H , Popovic A , Kovacic F , Brown G , Flick R , Hajighasemi M , Egorova O , Somody JC , Tchigvintsev D , Khusnutdinova A , Chernikova TN , Golyshina OV , Yakimov MM , Savchenko A , Golyshin PN , Jaeger KE , Yakunin AF
Ref : Applied Microbiology & Biotechnology , 99 :2165 , 2015
Abstract : Most of the Earth's biosphere is cold and is populated by cold-adapted microorganisms. To explore the natural enzyme diversity of these environments and identify new carboxylesterases, we have screened three marine metagenome gene libraries for esterase activity. The screens identified 23 unique active clones, from which five highly active esterases were selected for biochemical characterization. The purified metagenomic esterases exhibited high activity against alpha-naphthyl and p-nitrophenyl esters with different chain lengths. All five esterases retained high activity at 5 degrees C indicating that they are cold-adapted enzymes. The activity of MGS0010 increased more than two times in the presence of up to 3.5 M NaCl or KCl, whereas the other four metagenomic esterases were inhibited to various degrees by these salts. The purified enzymes showed different sensitivities to inhibition by solvents and detergents, and the activities of MGS0010, MGS0105 and MGS0109 were stimulated three to five times by the addition of glycerol. Screening of purified esterases against 89 monoester substrates revealed broad substrate profiles with a preference for different esters. The metagenomic esterases also hydrolyzed several polyester substrates including polylactic acid suggesting that they can be used for polyester depolymerization. Thus, esterases from marine metagenomes are cold-adapted enzymes exhibiting broad biochemical diversity reflecting the environmental conditions where they evolved.
ESTHER : Tchigvintsev_2015_Appl.Microbiol.Biotechnol_99_2165
PubMedSearch : Tchigvintsev_2015_Appl.Microbiol.Biotechnol_99_2165
PubMedID: 25194841

Title : Structure and activity of the cold-active and anion-activated carboxyl esterase OLEI01171 from the oil-degrading marine bacterium Oleispira antarctica - Lemak_2012_Biochem.J_445_193
Author(s) : Lemak S , Tchigvintsev A , Petit P , Flick R , Singer AU , Brown G , Evdokimova E , Egorova O , Gonzalez CF , Chernikova TN , Yakimov MM , Kube M , Reinhardt R , Golyshin PN , Savchenko A , Yakunin AF
Ref : Biochemical Journal , 445 :193 , 2012
Abstract : The uncharacterized alpha/beta-hydrolase protein OLEI01171 from the psychrophilic marine bacterium Oleispira antarctica belongs to the PF00756 family of putative esterases, which also includes human esterase D. In the present paper we show that purified recombinant OLEI01171 exhibits high esterase activity against the model esterase substrate alpha-naphthyl acetate at 5-30 degrees C with maximal activity at 15-20 degrees C. The esterase activity of OLEI01171 was stimulated 3-8-fold by the addition of chloride or several other anions (0.1-1.0 M). Compared with mesophilic PF00756 esterases, OLEI01171 exhibited a lower overall protein thermostability. Two crystal structures of OLEI01171 were solved at 1.75 and 2.1 A resolution and revealed a classical serine hydrolase catalytic triad and the presence of a chloride or bromide ion bound in the active site close to the catalytic Ser148. Both anions were found to co-ordinate a potential catalytic water molecule located in the vicinity of the catalytic triad His257. The results of the present study suggest that the bound anion perhaps contributes to the polarization of the catalytic water molecule and increases the rate of the hydrolysis of an acyl-enzyme intermediate. Alanine replacement mutagenesis of OLEI01171 identified ten amino acid residues important for esterase activity. The replacement of Asn225 by lysine had no significant effect on the activity or thermostability of OLEI01171, but resulted in a detectable increase of activity at 35-45 degrees C. The present study has provided insight into the molecular mechanisms of activity of a cold-active and anion-activated carboxyl esterase.
ESTHER : Lemak_2012_Biochem.J_445_193
PubMedSearch : Lemak_2012_Biochem.J_445_193
PubMedID: 22519667
Gene_locus related to this paper: olean-d0vwz4

Title : The genome of the model beetle and pest Tribolium castaneum - Richards_2008_Nature_452_949
Author(s) : Richards S , Gibbs RA , Weinstock GM , Brown SJ , Denell R , Beeman RW , Gibbs R , Bucher G , Friedrich M , Grimmelikhuijzen CJ , Klingler M , Lorenzen M , Roth S , Schroder R , Tautz D , Zdobnov EM , Muzny D , Attaway T , Bell S , Buhay CJ , Chandrabose MN , Chavez D , Clerk-Blankenburg KP , Cree A , Dao M , Davis C , Chacko J , Dinh H , Dugan-Rocha S , Fowler G , Garner TT , Garnes J , Gnirke A , Hawes A , Hernandez J , Hines S , Holder M , Hume J , Jhangiani SN , Joshi V , Khan ZM , Jackson L , Kovar C , Kowis A , Lee S , Lewis LR , Margolis J , Morgan M , Nazareth LV , Nguyen N , Okwuonu G , Parker D , Ruiz SJ , Santibanez J , Savard J , Scherer SE , Schneider B , Sodergren E , Vattahil S , Villasana D , White CS , Wright R , Park Y , Lord J , Oppert B , Brown S , Wang L , Weinstock G , Liu Y , Worley K , Elsik CG , Reese JT , Elhaik E , Landan G , Graur D , Arensburger P , Atkinson P , Beidler J , Demuth JP , Drury DW , Du YZ , Fujiwara H , Maselli V , Osanai M , Robertson HM , Tu Z , Wang JJ , Wang S , Song H , Zhang L , Werner D , Stanke M , Morgenstern B , Solovyev V , Kosarev P , Brown G , Chen HC , Ermolaeva O , Hlavina W , Kapustin Y , Kiryutin B , Kitts P , Maglott D , Pruitt K , Sapojnikov V , Souvorov A , Mackey AJ , Waterhouse RM , Wyder S , Kriventseva EV , Kadowaki T , Bork P , Aranda M , Bao R , Beermann A , Berns N , Bolognesi R , Bonneton F , Bopp D , Butts T , Chaumot A , Denell RE , Ferrier DE , Gordon CM , Jindra M , Lan Q , Lattorff HM , Laudet V , von Levetsow C , Liu Z , Lutz R , Lynch JA , da Fonseca RN , Posnien N , Reuter R , Schinko JB , Schmitt C , Schoppmeier M , Shippy TD , Simonnet F , Marques-Souza H , Tomoyasu Y , Trauner J , Van der Zee M , Vervoort M , Wittkopp N , Wimmer EA , Yang X , Jones AK , Sattelle DB , Ebert PR , Nelson D , Scott JG , Muthukrishnan S , Kramer KJ , Arakane Y , Zhu Q , Hogenkamp D , Dixit R , Jiang H , Zou Z , Marshall J , Elpidina E , Vinokurov K , Oppert C , Evans J , Lu Z , Zhao P , Sumathipala N , Altincicek B , Vilcinskas A , Williams M , Hultmark D , Hetru C , Hauser F , Cazzamali G , Williamson M , Li B , Tanaka Y , Predel R , Neupert S , Schachtner J , Verleyen P , Raible F , Walden KK , Angeli S , Foret S , Schuetz S , Maleszka R , Miller SC , Grossmann D
Ref : Nature , 452 :949 , 2008
Abstract : Tribolium castaneum is a member of the most species-rich eukaryotic order, a powerful model organism for the study of generalized insect development, and an important pest of stored agricultural products. We describe its genome sequence here. This omnivorous beetle has evolved the ability to interact with a diverse chemical environment, as shown by large expansions in odorant and gustatory receptors, as well as P450 and other detoxification enzymes. Development in Tribolium is more representative of other insects than is Drosophila, a fact reflected in gene content and function. For example, Tribolium has retained more ancestral genes involved in cell-cell communication than Drosophila, some being expressed in the growth zone crucial for axial elongation in short-germ development. Systemic RNA interference in T. castaneum functions differently from that in Caenorhabditis elegans, but nevertheless offers similar power for the elucidation of gene function and identification of targets for selective insect control.
ESTHER : Richards_2008_Nature_452_949
PubMedSearch : Richards_2008_Nature_452_949
PubMedID: 18362917
Gene_locus related to this paper: trica-ACHE1 , trica-ACHE2 , trica-d2a0g9 , trica-d2a0h0 , trica-d2a0w9 , trica-d2a0x0 , trica-d2a0x1 , trica-d2a0x3 , trica-d2a0x4.1 , trica-d2a0x4.2 , trica-d2a0x6 , trica-d2a2b8 , trica-d2a2h1 , trica-d2a3c3 , trica-d2a3g9 , trica-d2a5y5 , trica-d2a309 , trica-d2a514 , trica-d2a515 , trica-d2a516 , trica-d2a577 , trica-d2a578 , trica-d6w6x8 , trica-d6w7f9 , trica-d6w7h2 , trica-d6w8e7 , trica-d6w9c0 , trica-d6w855 , trica-d6wac8 , trica-d6wan4 , trica-d6wd50 , trica-d6wd73 , trica-d6wd74 , trica-A0A139WM97 , trica-d6wfu3 , trica-d6wgl2 , trica-d6wj57 , trica-d6wj59 , trica-d6wjs3 , trica-d6wl31 , trica-d6wnv1 , trica-d6wpl0 , trica-d6wqd6 , trica-d6wqr4 , trica-d6ws52 , trica-d6wsm0 , trica-d6wu38 , trica-d6wu39 , trica-d6wu40 , trica-d6wu41 , trica-d6wu44 , trica-d6wvk5 , trica-d6wvz7 , trica-d6wwu9 , trica-d6wwv0 , trica-d6wxz0 , trica-d6wyy1 , trica-d6wyy2 , trica-d6x0z2 , trica-d6x0z5 , trica-d6x0z6 , trica-d6x4b2 , trica-d6x4e8 , trica-d6x4e9 , trica-d6x197 , trica-d7eip7 , trica-d7eld3 , trica-d7us45 , trica-q5wm43 , trica-q5zex9 , trica-d6wie5 , trica-d6w7t0 , trica-d6x4h0 , trica-d6x4h1 , trica-a0a139wae8 , trica-a0a139wc96 , trica-d6x325 , trica-d2a4s2 , trica-d6wvw8

Title : Functional and structural characterization of four glutaminases from Escherichia coli and Bacillus subtilis - Brown_2008_Biochemistry_47_5724
Author(s) : Brown G , Singer A , Proudfoot M , Skarina T , Kim Y , Chang C , Dementieva I , Kuznetsova E , Gonzalez CF , Joachimiak A , Savchenko A , Yakunin AF
Ref : Biochemistry , 47 :5724 , 2008
Abstract : Glutaminases belong to the large superfamily of serine-dependent beta-lactamases and penicillin-binding proteins, and they catalyze the hydrolytic deamidation of L-glutamine to L-glutamate. In this work, we purified and biochemically characterized four predicted glutaminases from Escherichia coli (YbaS and YneH) and Bacillus subtilis (YlaM and YbgJ). The proteins demonstrated strict specificity to L-glutamine and did not hydrolyze D-glutamine or L-asparagine. In each organism, one glutaminase showed higher affinity to glutamine ( E. coli YbaS and B. subtilis YlaM; K m 7.3 and 7.6 mM, respectively) than the second glutaminase ( E. coli YneH and B. subtilis YbgJ; K m 27.6 and 30.6 mM, respectively). The crystal structures of the E. coli YbaS and the B. subtilis YbgJ revealed the presence of a classical beta-lactamase-like fold and conservation of several key catalytic residues of beta-lactamases (Ser74, Lys77, Asn126, Lys268, and Ser269 in YbgJ). Alanine replacement mutagenesis demonstrated that most of the conserved residues located in the putative glutaminase catalytic site are essential for activity. The crystal structure of the YbgJ complex with the glutaminase inhibitor 6-diazo-5-oxo- l-norleucine revealed the presence of a covalent bond between the inhibitor and the hydroxyl oxygen of Ser74, providing evidence that Ser74 is the primary catalytic nucleophile and that the glutaminase reaction proceeds through formation of an enzyme-glutamyl intermediate. Growth experiments with the E. coli glutaminase deletion strains revealed that YneH is involved in the assimilation of l-glutamine as a sole source of carbon and nitrogen and suggested that both glutaminases (YbaS and YneH) also contribute to acid resistance in E. coli.
ESTHER : Brown_2008_Biochemistry_47_5724
PubMedSearch : Brown_2008_Biochemistry_47_5724
PubMedID: 18459799

Title : Molecular basis of formaldehyde detoxification. Characterization of two S-formylglutathione hydrolases from Escherichia coli, FrmB and YeiG - Gonzalez_2006_J.Biol.Chem_281_14514
Author(s) : Gonzalez CF , Proudfoot M , Brown G , Korniyenko Y , Mori H , Savchenko AV , Yakunin AF
Ref : Journal of Biological Chemistry , 281 :14514 , 2006
Abstract : The Escherichia coli genes frmB (yaiM) and yeiG encode two uncharacterized proteins that share 54% sequence identity and contain a serine esterase motif. We demonstrated that purified FrmB and YeiG have high carboxylesterase activity against the model substrates, p-nitrophenyl esters of fatty acids (C2-C6) and alpha-naphthyl acetate. However, both proteins had the highest hydrolytic activity toward S-formylglutathione, an intermediate of the glutathione-dependent pathway of formaldehyde detoxification. With this substrate, both proteins had similar affinity (Km = 0.41-0.43 mM), but FrmB was almost 5 times more active. Alanine replacement mutagenesis of YeiG demonstrated that Ser145, Asp233, and His256 are absolutely required for activity, indicating that these residues represent a serine hydrolase catalytic triad in this protein and in other S-formylglutathione hydrolases. This was confirmed by inspecting the crystal structure of the Saccharomyces cerevisiae S-formylglutathione hydrolase YJG8 (Protein Data Bank code 1pv1), which has 45% sequence identity to YeiG. The structure revealed a canonical alpha/beta-hydrolase fold and a classical serine hydrolase catalytic triad (Ser161, His276, Asp241). In E. coli cells, the expression of frmB was stimulated 45-75 times by the addition of formaldehyde to the growth medium, whereas YeiG was found to be a constitutive enzyme. The simultaneous deletion of both frmB and yeiG genes was required to increase the sensitivity of the growth of E. coli cells to formaldehyde, suggesting that both FrmB and YeiG contribute to the detoxification of formaldehyde. Thus, FrmB and YeiG are S-formylglutathione hydrolases with a Ser-His-Asp catalytic triad involved in the detoxification of formaldehyde in E. coli.
ESTHER : Gonzalez_2006_J.Biol.Chem_281_14514
PubMedSearch : Gonzalez_2006_J.Biol.Chem_281_14514
PubMedID: 16567800
Gene_locus related to this paper: ecoli-yaim , ecoli-yeiG