Flick R

References (8)

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 : 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 : Pressure adaptation is linked to thermal adaptation in salt-saturated marine habitats - Alcaide_2015_Environ.Microbiol_17_332
Author(s) : Alcaide M , Stogios PJ , Lafraya A , Tchigvintsev A , Flick R , Bargiela R , Chernikova TN , Reva ON , Hai T , Leggewie CC , Katzke N , La Cono V , Matesanz R , Jebbar M , Jaeger KE , Yakimov MM , Yakunin AF , Golyshin PN , Golyshina OV , Savchenko A , Ferrer M
Ref : Environ Microbiol , 17 :332 , 2015
Abstract : The present study provides a deeper view of protein functionality as a function of temperature, salt and pressure in deep-sea habitats. A set of eight different enzymes from five distinct deep-sea (3040-4908 m depth), moderately warm (14.0-16.5 degrees C) biotopes, characterized by a wide range of salinities (39-348 practical salinity units), were investigated for this purpose. An enzyme from a 'superficial' marine hydrothermal habitat (65 degrees C) was isolated and characterized for comparative purposes. We report here the first experimental evidence suggesting that in salt-saturated deep-sea habitats, the adaptation to high pressure is linked to high thermal resistance (P value = 0.0036). Salinity might therefore increase the temperature window for enzyme activity, and possibly microbial growth, in deep-sea habitats. As an example, Lake Medee, the largest hypersaline deep-sea anoxic lake of the Eastern Mediterranean Sea, where the water temperature is never higher than 16 degrees C, was shown to contain halopiezophilic-like enzymes that are most active at 70 degrees C and with denaturing temperatures of 71.4 degrees C. The determination of the crystal structures of five proteins revealed unknown molecular mechanisms involved in protein adaptation to poly-extremes as well as distinct active site architectures and substrate preferences relative to other structurally characterized enzymes.
ESTHER : Alcaide_2015_Environ.Microbiol_17_332
PubMedSearch : Alcaide_2015_Environ.Microbiol_17_332
PubMedID: 25330254
Gene_locus related to this paper: 9alte-MGS-MT1 , 9bact-MGS-M1 , 9bact-MGS-M2 , 9bact-a0a0b5kns5

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 : Genome sequence and functional genomic analysis of the oil-degrading bacterium Oleispira antarctica - Kube_2013_Nat.Commun_4_2156
Author(s) : Kube M , Chernikova TN , Al-Ramahi Y , Beloqui A , Lopez-Cortez N , Guazzaroni ME , Heipieper HJ , Klages S , Kotsyurbenko OR , Langer I , Nechitaylo TY , Lunsdorf H , Fernandez M , Juarez S , Ciordia S , Singer A , Kagan O , Egorova O , Petit PA , Stogios P , Kim Y , Tchigvintsev A , Flick R , Denaro R , Genovese M , Albar JP , Reva ON , Martinez-Gomariz M , Tran H , Ferrer M , Savchenko A , Yakunin AF , Yakimov MM , Golyshina OV , Reinhardt R , Golyshin PN
Ref : Nat Commun , 4 :2156 , 2013
Abstract : Ubiquitous bacteria from the genus Oleispira drive oil degradation in the largest environment on Earth, the cold and deep sea. Here we report the genome sequence of Oleispira antarctica and show that compared with Alcanivorax borkumensis--the paradigm of mesophilic hydrocarbonoclastic bacteria--O. antarctica has a larger genome that has witnessed massive gene-transfer events. We identify an array of alkane monooxygenases, osmoprotectants, siderophores and micronutrient-scavenging pathways. We also show that at low temperatures, the main protein-folding machine Cpn60 functions as a single heptameric barrel that uses larger proteins as substrates compared with the classical double-barrel structure observed at higher temperatures. With 11 protein crystal structures, we further report the largest set of structures from one psychrotolerant organism. The most common structural feature is an increased content of surface-exposed negatively charged residues compared to their mesophilic counterparts. Our findings are relevant in the context of microbial cold-adaptation mechanisms and the development of strategies for oil-spill mitigation in cold environments.
ESTHER : Kube_2013_Nat.Commun_4_2156
PubMedSearch : Kube_2013_Nat.Commun_4_2156
PubMedID: 23877221
Gene_locus related to this paper: olean-olei00960 , olean-r4ym14 , olean-r4yv64 , olean-r4ys13

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