Joachimiak A

References (9)

Title : Data collection from crystals grown in microfluidic droplets - Babnigg_2022_Acta.Crystallogr.D.Struct.Biol_78_997
Author(s) : Babnigg G , Sherrell D , Kim Y , Johnson JL , Nocek B , Tan K , Axford D , Li H , Bigelow L , Welk L , Endres M , Owen RL , Joachimiak A
Ref : Acta Crystallographica D Struct Biol , 78 :997 , 2022
Abstract : Protein crystals grown in microfluidic droplets have been shown to be an effective and robust platform for storage, transport and serial crystallography data collection with a minimal impact on diffraction quality. Single macromolecular microcrystals grown in nanolitre-sized droplets allow the very efficient use of protein samples and can produce large quantities of high-quality samples for data collection. However, there are challenges not only in growing crystals in microfluidic droplets, but also in delivering the droplets into X-ray beams, including the physical arrangement, beamline and timing constraints and ease of use. Here, the crystallization of two human gut microbial hydrolases in microfluidic droplets is described: a sample-transport and data-collection approach that is inexpensive, is convenient, requires small amounts of protein and is forgiving. It is shown that crystals can be grown in 50-500pl droplets when the crystallization conditions are compatible with the droplet environment. Local and remote data-collection methods are described and it is shown that crystals grown in microfluidics droplets and housed as an emulsion in an Eppendorf tube can be shipped from the US to the UK using a FedEx envelope, and data can be collected successfully. Details of how crystals were delivered to the X-ray beam by depositing an emulsion of droplets onto a silicon fixed-target serial device are provided. After three months of storage at 4 degreesC, the crystals endured and diffracted well, showing only a slight decrease in diffracting power, demonstrating a suitable way to grow crystals, and to store and collect the droplets with crystals for data collection. This sample-delivery and data-collection strategy allows crystal droplets to be shipped and set aside until beamtime is available.
ESTHER : Babnigg_2022_Acta.Crystallogr.D.Struct.Biol_78_997
PubMedSearch : Babnigg_2022_Acta.Crystallogr.D.Struct.Biol_78_997
PubMedID: 35916224

Title : Target highlights in CASP14: Analysis of models by structure providers - Alexander_2021_Proteins__
Author(s) : Alexander LT , Lepore R , Kryshtafovych A , Adamopoulos A , Alahuhta M , Arvin AM , Bomble YJ , Bottcher B , Breyton C , Chiarini V , Chinnam NB , Chiu W , Fidelis K , Grinter R , Gupta GD , Hartmann MD , Hayes CS , Heidebrecht T , Ilari A , Joachimiak A , Kim Y , Linares R , Lovering AL , Lunin VV , Lupas AN , Makbul C , Michalska K , Moult J , Mukherjee PK , Nutt WS , Oliver SL , Perrakis A , Stols L , Tainer JA , Topf M , Tsutakawa SE , Valdivia-Delgado M , Schwede T
Ref : Proteins , : , 2021
Abstract : The biological and functional significance of selected Critical Assessment of Techniques for Protein Structure Prediction 14 (CASP14) targets are described by the authors of the structures. The authors highlight the most relevant features of the target proteins and discuss how well these features were reproduced in the respective submitted predictions. The overall ability to predict three-dimensional structures of proteins has improved remarkably in CASP14, and many difficult targets were modeled with impressive accuracy. For the first time in the history of CASP, the experimentalists not only highlighted that computational models can accurately reproduce the most critical structural features observed in their targets, but also envisaged that models could serve as a guidance for further studies of biologically-relevant properties of proteins.
ESTHER : Alexander_2021_Proteins__
PubMedSearch : Alexander_2021_Proteins__
PubMedID: 34561912

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 : New aminopeptidase from microbial dark matter archaeon - Michalska_2015_FASEB.J_29_4071
Author(s) : Michalska K , Steen AD , Chhor G , Endres M , Webber AT , Bird J , Lloyd KG , Joachimiak A
Ref : FASEB Journal , 29 :4071 , 2015
Abstract : Marine sediments host a large population of diverse, heterotrophic, uncultured microorganisms with unknown physiologies that control carbon flow through organic matter decomposition. Recently, single-cell genomics uncovered new key players in these processes, such as the miscellaneous crenarchaeotal group. These widespread archaea encode putative intra- and extracellular proteases for the degradation of detrital proteins present in sediments. Here, we show that one of these enzymes is a self-compartmentalizing tetrameric aminopeptidase with a preference for cysteine and hydrophobic residues at the N terminus of the hydrolyzed peptide. The ability to perform detailed characterizations of enzymes from native subsurface microorganisms, without requiring that those organisms first be grown in pure culture, holds great promise for understanding key carbon transformations in the environment as well as identifying new enzymes for biomedical and biotechnological applications.
ESTHER : Michalska_2015_FASEB.J_29_4071
PubMedSearch : Michalska_2015_FASEB.J_29_4071
PubMedID: 26062601
Gene_locus related to this paper: 9arch-m7tve7

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 : Harvesting the high-hanging fruit: the structure of the YdeN gene product from Bacillus subtilis at 1.8 angstroms resolution - Janda_2004_Acta.Crystallogr.D.Biol.Crystallogr_60_1101
Author(s) : Janda I , Devedjiev Y , Cooper D , Chruszcz M , Derewenda U , Gabrys A , Minor W , Joachimiak A , Derewenda ZS
Ref : Acta Crystallographica D Biol Crystallogr , 60 :1101 , 2004
Abstract : High-throughput (HT) protein crystallography is severely impeded by the relatively low success rate of protein crystallization. Proteins whose structures are not solved in the HT pipeline owing to attrition in any phase of the project are referred to as the high-hanging fruit, in contrast to those proteins that yielded good-quality crystals and crystal structures, which are referred to as low-hanging fruit. It has previously been shown that proteins that do not crystallize in the wild-type form can have their surfaces engineered by site-directed mutagenesis in order to create patches of low conformational entropy that are conducive to forming intermolecular interactions. The application of this method to selected proteins from the Bacillus subtilis genome which failed to crystallize in the HT mode is now reported. In this paper, the crystal structure of the product of the YdeN gene is reported. Of three prepared double mutants, i.e. E124A/K127A, E167A/E169A and K88A/Q89A, the latter gave high-quality crystals and the crystal structure was solved by SAD at 1.8 angstroms resolution. The protein is a canonical alpha/beta hydrolase, with an active site that is accessible to solvent.
ESTHER : Janda_2004_Acta.Crystallogr.D.Biol.Crystallogr_60_1101
PubMedSearch : Janda_2004_Acta.Crystallogr.D.Biol.Crystallogr_60_1101
PubMedID: 15159570
Gene_locus related to this paper: bacsu-YDEN

Title : Integrating structure, bioinformatics, and enzymology to discover function: BioH, a new carboxylesterase from Escherichia coli - Sanishvili_2003_J.Biol.Chem_278_26039
Author(s) : Sanishvili R , Yakunin AF , Laskowski RA , Skarina T , Evdokimova E , Doherty-Kirby A , Lajoie GA , Thornton JM , Arrowsmith CH , Savchenko A , Joachimiak A , Edwards AM
Ref : Journal of Biological Chemistry , 278 :26039 , 2003
Abstract : Structural proteomics projects are generating three-dimensional structures of novel, uncharacterized proteins at an increasing rate. However, structure alone is often insufficient to deduce the specific biochemical function of a protein. Here we determined the function for a protein using a strategy that integrates structural and bioinformatics data with parallel experimental screening for enzymatic activity. BioH is involved in biotin biosynthesis in Escherichia coli and had no previously known biochemical function. The crystal structure of BioH was determined at 1.7 A resolution. An automated procedure was used to compare the structure of BioH with structural templates from a variety of different enzyme active sites. This screen identified a catalytic triad (Ser82, His235, and Asp207) with a configuration similar to that of the catalytic triad of hydrolases. Analysis of BioH with a panel of hydrolase assays revealed a carboxylesterase activity with a preference for short acyl chain substrates. The combined use of structural bioinformatics with experimental screens for detecting enzyme activity could greatly enhance the rate at which function is determined from structure.
ESTHER : Sanishvili_2003_J.Biol.Chem_278_26039
PubMedSearch : Sanishvili_2003_J.Biol.Chem_278_26039
PubMedID: 12732651
Gene_locus related to this paper: ecoli-bioh