scholarly journals Pyrethroid Carboxylesterase PytH from Sphingobium faniae JZ-2: Structure and Catalytic Mechanism

2020 ◽  
Vol 86 (12) ◽  
Author(s):  
Dongqing Xu ◽  
Yanyan Gao ◽  
Bo Sun ◽  
Tingting Ran ◽  
Liangping Zeng ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Catalytic Mechanism ◽  
Hydrophobic Pocket ◽  
Core Domain ◽  
Content Type ◽  
Sequence Identity ◽  
Hydrolase Fold ◽  
Wide Range ◽  
Pyrethroid Pesticides ◽  
Lid Domain

ABSTRACT Carboxylesterase PytH, isolated from the pyrethroid-degrading bacterium Sphingobium faniae JZ-2, could rapidly hydrolyze the ester bond of a wide range of pyrethroid pesticides, including permethrin, fenpropathrin, cypermethrin, fenvalerate, deltamethrin, cyhalothrin, and bifenthrin. To elucidate the catalytic mechanism of PytH, we report here the crystal structures of PytH with bifenthrin (BIF) and phenylmethylsulfonyl fluoride (PMSF) and two PytH mutants. Though PytH shares low sequence identity with reported α/β-hydrolase fold proteins, the typical triad catalytic center with Ser-His-Asp triad (Ser78, His230, and Asp202) is present and vital for the hydrolase activity. However, no contact was found between Ser78 and His230 in the structures we solved, which may be due to the fact that the PytH structures we determined are in their inactive or low-activity forms. The structure of PytH is composed of a core domain and a lid domain; some hydrophobic amino acid residues surrounding the substrate from both domains form a deeper and wider hydrophobic pocket than its homologous structures. This indicates that the larger hydrophobic pocket makes PytH fit for its larger substrate binding; both lid and core domains are involved in substrate binding, and the lid domain-induced core domain movement may make the active center correctly positioned with substrates. IMPORTANCE Pyrethroid pesticides are widely applied in agriculture and household; however, extensive use of these pesticides also causes serious environmental and health problems. The hydrolysis of pyrethroids by carboxylesterases is the major pathway of microbial degradation of pyrethroids, but the structure of carboxylesterases and its catalytic mechanism are still unknown. Carboxylesterase PytH from Sphingobium faniae JZ-2 could effectively hydrolyze a wide range of pyrethroid pesticides. The crystal structures of PytH are solved in this study. This showed that PytH belongs to the α/β-hydrolase fold proteins with typical catalytic Ser-His-Asp triad, though PytH has a low sequence identity (about 20%) with them. The special large hydrophobic binding pocket enabled PytH to bind bigger pyrethroid family substrates. Our structures shed light on the substrate selectivity and the future application of PytH and deepen our understanding of α/β-hydrolase members.

scholarly journals Structure of GPN-Loop GTPase Npa3 and Implications for RNA Polymerase II Assembly

2015 ◽  
Vol 36 (5) ◽  
pp. 820-831 ◽  
Author(s):  
Jürgen Niesser ◽  
Felix R. Wagner ◽  
Dirk Kostrewa ◽  
Wolfgang Mühlbacher ◽  
Patrick Cramer
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Catalytic Mechanism ◽  
Protein Complexes ◽  
Hydrophobic Pocket ◽  
Pol Ii ◽  
Content Type ◽  
Open Conformation ◽  
Peptide Release ◽  
Hydrophobic Peptide

Biogenesis of the 12-subunit RNA polymerase II (Pol II) transcription complex requires so-called GPN-loop GTPases, but the function of these enzymes is unknown. Here we report the first crystal structure of a eukaryotic GPN-loop GTPase, theSaccharomyces cerevisiaeenzyme Npa3 (a homolog of human GPN1, also called RPAP4, XAB1, and MBDin), and analyze its catalytic mechanism. The enzyme was trapped in a GDP-bound closed conformation and in a novel GTP analog-bound open conformation displaying a conserved hydrophobic pocket distant from the active site. We show that Npa3 has chaperone activity and interacts with hydrophobic peptide regions of Pol II subunits that form interfaces in the assembled Pol II complex. Biochemical results are consistent with a model that the hydrophobic pocket binds peptides and that this can allosterically stimulate GTPase activity and subsequent peptide release. These results suggest that GPN-loop GTPases are assembly chaperones for Pol II and other protein complexes.

Crystal structures of Cg1458 reveal a catalytic lid domain and a common catalytic mechanism for the FAH family

2012 ◽  
Vol 449 (1) ◽  
pp. 51-60 ◽  
Author(s):  
Tingting Ran ◽  
Yanyan Gao ◽  
May Marsh ◽  
Wenjun Zhu ◽  
Meitian Wang ◽  
...  
Keyword(s):  
Glutamic Acid ◽  
Crystal Structures ◽  
Sequence Alignment ◽  
Large Scale ◽  
Catalytic Mechanism ◽  
Enzymatic Catalysis ◽  
Catalytic Triad ◽  
Sequence Motifs ◽  
Fumarylacetoacetate Hydrolase ◽  
Lid Domain

Cg1458 was recently characterized as a novel soluble oxaloacetate decarboxylase. However, sequence alignment identified that Cg1458 has no similarity with other oxaloacetate decarboxylases and instead belongs to the FAH (fumarylacetoacetate hydrolase) family. Differences in the function of Cg1458 and other FAH proteins may suggest a different catalytic mechanism. To help elucidate the catalytic mechanism of Cg1458, crystal structures of Cg1458 in both the open and closed conformations have been determined for the first time up to a resolution of 1.9 Å (1 Å=0.1 nm) and 2.0 Å respectively. Comparison of both structures and detailed biochemical studies confirmed the presence of a catalytic lid domain which is missing in the native enzyme structure. In this lid domain, a glutamic acid–histidine dyad was found to be critical in mediating enzymatic catalysis. On the basis of structural modelling and comparison, as well as large-scale sequence alignment studies, we further determined that the catalytic mechanism of Cg1458 is actually through a glutamic acid–histidine–water triad, and this catalytic triad is common among FAH family proteins that catalyse the cleavage of the C–C bond of the substrate. Two sequence motifs, HxxE and Hxx…xxE have been identified as the basis for this mechanism.

scholarly journals Evidence for substrate-assisted catalysis in N-acetylphosphoglucosamine mutase

2018 ◽  
Vol 475 (15) ◽  
pp. 2547-2557
Author(s):  
Olawale G. Raimi ◽  
Ramon Hurtado-Guerrero ◽  
Daan M.F. van Aalten
Keyword(s):  
Crystal Structures ◽  
Biosynthetic Pathway ◽  
Catalytic Mechanism ◽  
Phosphate Group ◽  
Structural Level ◽  
Hexosamine Biosynthetic Pathway ◽  
Wide Range

N-acetylphosphoglucosamine mutase (AGM1) is a key component of the hexosamine biosynthetic pathway that produces UDP-GlcNAc, an essential precursor for a wide range of glycans in eukaryotes. AGM belongs to the α-d-phosphohexomutase metalloenzyme superfamily and catalyzes the interconversion of N-acetylglucosamine-6-phosphate (GlcNAc-6P) to N-acetylglucosamine-1-phosphate (GlcNAc-1P) through N-acetylglucosamine-1,6-bisphosphate (GlcNAc-1,6-bisP) as the catalytic intermediate. Although there is an understanding of the phosphoserine-dependent catalytic mechanism at enzymatic and structural level, the identity of the requisite catalytic base in AGM1/phosphoglucomutases is as yet unknown. Here, we present crystal structures of a Michaelis complex of AGM1 with GlcNAc-6P and Mg2+, and a complex of the inactive Ser69Ala mutant together with glucose-1,6-bisphosphate (Glc-1,6-bisP) that represents key snapshots along the reaction co-ordinate. Together with mutagenesis, these structures reveal that the phosphate group of the hexose-1,6-bisP intermediate may act as the catalytic base.

Crystal structures and catalytic mechanism of theC-methyltransferase Coq5 provide insights into a key step of the yeast coenzyme Q synthesis pathway

2014 ◽  
Vol 70 (8) ◽  
pp. 2085-2092 ◽  
Author(s):  
Ya-Nan Dai ◽  
Kang Zhou ◽  
Dong-Dong Cao ◽  
Yong-Liang Jiang ◽  
Fei Meng ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Conformational Changes ◽  
Catalytic Mechanism ◽  
Coenzyme Q ◽  
Computational Simulation ◽  
Binding Pocket ◽  
Multiple Sequence ◽  
Core Domain ◽  
Dimeric Interface ◽  
Adenosyl Methionine

Saccharomyces cerevisiaeCoq5 is anS-adenosyl methionine (SAM)-dependent methyltransferase (SAM-MTase) that catalyzes the onlyC-methylation step in the coenzyme Q (CoQ) biosynthesis pathway, in which 2-methoxy-6-polyprenyl-1,4-benzoquinone (DDMQH2) is converted to 2-methoxy-5-methyl-6-polyprenyl-1,4-benzoquinone (DMQH2). Crystal structures of Coq5 were determined in the apo form (Coq5-apo) at 2.2 Å resolution and in the SAM-bound form (Coq5-SAM) at 2.4 Å resolution, representing the first pair of structures for the yeast CoQ biosynthetic enzymes. Coq5 displays a typical class I SAM-MTase structure with two minor variations beyond the core domain, both of which are considered to participate in dimerization and/or substrate recognition. Slight conformational changes at the active-site pocket were observed upon binding of SAM. Structure-based computational simulation using an analogue of DDMQH2enabled us to identify the binding pocket and entrance tunnel of the substrate. Multiple-sequence alignment showed that the residues contributing to the dimeric interface and the SAM- and DDMQH2-binding sites are highly conserved in Coq5 and homologues from diverse species. A putative catalytic mechanism of Coq5 was proposed in which Arg201 acts as a general base to initiate catalysis with the help of a water molecule.

scholarly journals Cloning of a Novel Pyrethroid-Hydrolyzing Carboxylesterase Gene from Sphingobium sp. Strain JZ-1 and Characterization of the Gene Product

2009 ◽  
Vol 75 (17) ◽  
pp. 5496-5500 ◽  
Author(s):  
Bao-zhan Wang ◽  
Peng Guo ◽  
Bao-jian Hang ◽  
Lian Li ◽  
Jian He ◽  
...  
Keyword(s):  
Short Chain Fatty Acids ◽  
Nitrilotriacetic Acid ◽  
Reading Frame ◽  
Hydrolase Fold ◽  
Wide Range ◽  
Pyrethroid Pesticides ◽  
Novel Esterase ◽  
Esterase Gene ◽  
Monomeric Structure

ABSTRACT A novel esterase gene, pytH, encoding a pyrethroid-hydrolyzing carboxylesterase was cloned from Sphingobium sp. strain JZ-1. The gene contained an open reading frame of 840 bp. Sequence identity searches revealed that the deduced enzyme shared the highest similarity with many α/β-hydrolase fold proteins (20 to 24% identities). PytH was expressed in Escherichia coli BL21 and purified using Ni-nitrilotriacetic acid affinity chromatography. It was a monomeric structure with a molecular mass of approximately 31 kDa and a pI of 4.85. PytH was able to transform p-nitrophenyl esters of short-chain fatty acids and a wide range of pyrethroid pesticides, and isomer selectivity was not observed. No cofactors were required for enzyme activity.

scholarly journals NADP+-Preferring d-Lactate Dehydrogenase from Sporolactobacillus inulinus

2015 ◽  
Vol 81 (18) ◽  
pp. 6294-6301 ◽  
Author(s):  
Lingfeng Zhu ◽  
Xiaoling Xu ◽  
Limin Wang ◽  
Hui Dong ◽  
Bo Yu ◽  
...  
Keyword(s):  
Lactate Dehydrogenase ◽  
Catalytic Mechanism ◽  
High Specificity ◽  
Biochemical Properties ◽  
Enzymatic System ◽  
Content Type ◽  
Wide Range ◽  
Lactate Dehydrogenases

ABSTRACTHydroxy acid dehydrogenases, includingl- andd-lactate dehydrogenases (L-LDH and D-LDH), are responsible for the stereospecific conversion of 2-keto acids to 2-hydroxyacids and extensively used in a wide range of biotechnological applications. A common feature of LDHs is their high specificity for NAD+as a cofactor. An LDH that could effectively use NADPH as a coenzyme could be an alternative enzymatic system for regeneration of the oxidized, phosphorylated cofactor. In this study, ad-lactate dehydrogenase from aSporolactobacillus inulinusstrain was found to use both NADH and NADPH with high efficiencies and with a preference for NADPH as its coenzyme, which is different from the coenzyme utilization of all previously reported LDHs. The biochemical properties of the D-LDH enzyme were determined by X-ray crystal structural characterization andin vivoandin vitroenzymatic activity analyses. The residue Asn174was demonstrated to be critical for NADPH utilization. Characterization of the biochemical properties of this enzyme will contribute to understanding of the catalytic mechanism and provide referential information for shifting the coenzyme utilization specificity of 2-hydroxyacid dehydrogenases.

scholarly journals Transfer of Plasmid DNA to Clinical Coagulase-Negative Staphylococcal Pathogens by Using a Unique Bacteriophage

2015 ◽  
Vol 81 (7) ◽  
pp. 2481-2488 ◽  
Author(s):  
Volker Winstel ◽  
Petra Kühner ◽  
Bernhard Krismer ◽  
Andreas Peschel ◽  
Holger Rohde
Keyword(s):  
Plasmid Dna ◽  
Genetic Manipulation ◽  
Current Knowledge ◽  
Virulence Determinants ◽  
Coagulase Negative Staphylococci ◽  
Reliable Technique ◽  
Content Type ◽  
Research Activities ◽  
Wide Range ◽  
Genetic Barriers

ABSTRACTGenetic manipulation of emerging bacterial pathogens, such as coagulase-negative staphylococci (CoNS), is a major hurdle in clinical and basic microbiological research. Strong genetic barriers, such as restriction modification systems or clustered regularly interspaced short palindromic repeats (CRISPR), usually interfere with available techniques for DNA transformation and therefore complicate manipulation of CoNS or render it impossible. Thus, current knowledge of pathogenicity and virulence determinants of CoNS is very limited. Here, a rapid, efficient, and highly reliable technique is presented to transfer plasmid DNA essential for genetic engineering to important CoNS pathogens from a uniqueStaphylococcus aureusstrain via a specificS. aureusbacteriophage, Φ187. Even strains refractory to electroporation can be transduced by this technique once donor and recipient strains share similar Φ187 receptor properties. As a proof of principle, this technique was used to delete the alternative transcription factor sigma B (SigB) via allelic replacement in nasal and clinicalStaphylococcus epidermidisisolates at high efficiencies. The described approach will allow the genetic manipulation of a wide range of CoNS pathogens and might inspire research activities to manipulate other important pathogens in a similar fashion.

scholarly journals Use of Proteins Identified through a Functional Genomic Screen To Develop a Protein Subunit Vaccine That Provides Significant Protection against Virulent Streptococcus suis in Pigs

2017 ◽  
Vol 86 (3) ◽  
Author(s):  
Susan L. Brockmeier ◽  
Crystal L. Loving ◽  
Tracy L. Nicholson ◽  
Jinhong Wang ◽  
Sarah E. Peters ◽  
...  
Keyword(s):  
Immune Response ◽  
Vaccine Trial ◽  
Streptococcus Suis ◽  
Protein Subunit ◽  
Content Type ◽  
Degree Of Protection ◽  
Wide Range ◽  
Associated Proteins ◽  
Clinical Protection ◽  
Cellular Immune

ABSTRACT Streptococcus suis is a bacterium that is commonly carried in the respiratory tract and that is also one of the most important invasive pathogens of swine, commonly causing meningitis, arthritis, and septicemia. Due to the existence of many serotypes and a wide range of immune evasion capabilities, efficacious vaccines are not readily available. The selection of S. suis protein candidates for inclusion in a vaccine was accomplished by identifying fitness genes through a functional genomics screen and selecting conserved predicted surface-associated proteins. Five candidate proteins were selected for evaluation in a vaccine trial and administered both intranasally and intramuscularly with one of two different adjuvant formulations. Clinical protection was evaluated by subsequent intranasal challenge with virulent S. suis . While subunit vaccination with the S. suis proteins induced IgG antibodies to each individual protein and a cellular immune response to the pool of proteins and provided substantial protection from challenge with virulent S. suis , the immune response elicited and the degree of protection were dependent on the parenteral adjuvant given. Subunit vaccination induced IgG reactive against different S. suis serotypes, indicating a potential for cross protection.

scholarly journals Structural and functional insights into esterase-mediated macrolide resistance

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Michał Zieliński ◽  
Jaeok Park ◽  
Barry Sleno ◽  
Albert M. Berghuis
Keyword(s):  
Active Site ◽  
Pathogenic Bacteria ◽  
Catalytic Mechanism ◽  
Three Dimensional ◽  
Resistance Mechanisms ◽  
Docking Studies ◽  
Macrolide Resistance ◽  
Flexible Docking ◽  
Sequence Identity ◽  
Substrate Spectrum

AbstractMacrolides are a class of antibiotics widely used in both medicine and agriculture. Unsurprisingly, as a consequence of their exensive usage a plethora of resistance mechanisms have been encountered in pathogenic bacteria. One of these resistance mechanisms entails the enzymatic cleavage of the macrolides’ macrolactone ring by erythromycin esterases (Eres). The most frequently identified Ere enzyme is EreA, which confers resistance to the majority of clinically used macrolides. Despite the role Eres play in macrolide resistance, research into this family enzymes has been sparse. Here, we report the first three-dimensional structures of an erythromycin esterase, EreC. EreC is an extremely close homologue of EreA, displaying more than 90% sequence identity. Two structures of this enzyme, in conjunction with in silico flexible docking studies and previously reported mutagenesis data allowed for the proposal of a detailed catalytic mechanism for the Ere family of enzymes, labeling them as metal-independent hydrolases. Also presented are substrate spectrum assays for different members of the Ere family. The results from these assays together with an examination of residue conservation for the macrolide binding site in Eres, suggests two distinct active site archetypes within the Ere enzyme family.

scholarly journals Nano-Crystallization of Ln-Fluoride Crystals in Glass-Ceramics via Inducing of Yb3+ for Efficient Near-Infrared Upconversion Luminescence of Tm3+

Nanomaterials ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 1033
Author(s):  
Jianfeng Li ◽  
Yi Long ◽  
Qichao Zhao ◽  
Shupei Zheng ◽  
Zaijin Fang ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Near Infrared ◽  
Upconversion Luminescence ◽  
Glass Ceramics ◽  
Upconversion Emission ◽  
Ceramic Composites ◽  
Wide Range ◽  
Transmission Window ◽  
Optical Applications ◽  
Upconversion Emissions

Transparent glass-ceramic composites embedded with Ln-fluoride nanocrystals are prepared in this work to enhance the upconversion luminescence of Tm3+. The crystalline phases, microstructures, and photoluminescence properties of samples are carefully investigated. KYb3F10 nanocrystals are proved to controllably precipitate in the glass-ceramics via the inducing of Yb3+ when the doping concentration varies from 0.5 to 1.5 mol%. Pure near-infrared upconversion emissions are observed and the emission intensities are enhanced in the glass-ceramics as compared to in the precursor glass due to the incorporation of Tm3+ into the KYb3F10 crystal structures via substitutions for Yb3+. Furthermore, KYb2F7 crystals are also nano-crystallized in the glass-ceramics when the Yb3+ concentration exceeds 2.0 mol%. The upconversion emission intensity of Tm3+ is further enhanced by seven times as Tm3+ enters the lattice sites of pure KYb2F7 nanocrystals. The designed glass ceramics provide efficient gain materials for optical applications in the biological transmission window. Moreover, the controllable nano-crystallization strategy induced by Yb3+ opens a new way for engineering a wide range of functional nanomaterials with effective incorporation of Ln3+ ions into fluoride crystal structures.

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