Structure and mechanism of the chalcogen-detoxifying protein TehB from Escherichia coli

2011 ◽  
Vol 435 (1) ◽  
pp. 85-91 ◽  
Author(s):  
Hassanul G. Choudhury ◽  
Alexander D. Cameron ◽  
So Iwata ◽  
Konstantinos Beis
Keyword(s):  
Escherichia Coli ◽  
Reaction Mechanism ◽  
Active Site ◽  
Kinetic Data ◽  
Protein Analysis ◽  
Nucleophilic Attack ◽  
Conserved Residues ◽  
Low Levels ◽  
Living Organisms ◽  
Derivatives Of

The oxyanion derivatives of the chalcogens tellurium and selenium are toxic to living organisms even at very low levels. Bacteria have developed mechanisms to overcome their toxicity by methylating them. The structure of TehB from Escherichia coli has been determined in the presence of the cofactor analogues SAH (S-adenosylhomocysteine) and sinefungin (a non-hydrolysable form of S-adenosyl-L-methionine) at 1.48 Å (1 Å=0.1 nm) and 1.9 Å respectively. Interestingly, our kinetic data show that TehB does not discriminate between selenium or tellurite oxyanions, making it a very powerful detoxifying protein. Analysis of the active site has identified three conserved residues that are capable of binding and orientating the metals for nucleophilic attack: His176, Arg177 and Arg184. Mutagenesis studies revealed that the H176A and R184A mutants retained most of their activity, whereas the R177A mutant had 65% of its activity abolished. Based on the structure and kinetic data we propose an SN2 nucleophilic attack reaction mechanism. These data provide the first molecular understanding of the detoxification of chalcogens by bacteria.

scholarly journals Effect of mutations in the transmethylase and dehydrogenase/chelatase domains of sirohaem synthase (CysG) on sirohaem and cobalamin biosynthesis

1998 ◽  
Vol 330 (1) ◽  
pp. 121-129 ◽  
Author(s):  
C. Sarah WOODCOCK ◽  
Evelyne RAUX ◽  
Florence LEVILLAYER ◽  
Claude THERMES ◽  
Alain RAMBACH ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Catalytic Activity ◽  
Catalytic Domain ◽  
Conserved Residues ◽  
Pseudomonas Denitrificans ◽  
E Coli ◽  
Cobalamin Biosynthesis ◽  
N Terminus ◽  
Low Levels ◽  
Cobyric Acid

The Escherichia coli CysG protein (sirohaem synthase) catalyses four separate reactions that are required for the transformation of uroporphyrinogen III into sirohaem, initially two S-adenosyl-L-methionine-dependent transmethylations at positions 2 and 7, mediated through the C-terminal, or CysGA, catalytic domain of the protein, and subsequently a ferrochelation and dehydrogenation, mediated through the N-terminal, or CysGB, catalytic domain of the enzyme. This report describes how the deletion of the NAD+-binding site of CysG, located within the first 35 residues of the N-terminus, is detrimental to the activity of CysGB but does not affect the catalytic activity of CysGA, whereas the mutation of a number of phylogenetically conserved residues within CysGA is detrimental to the transmethylation reaction but does not affect the activity of CysGB. Further studies have shown that CysGB is not essential for cobalamin biosynthesis because the presence of the Salmonella typhimurium CobI operon with either cysGA or the Pseudomonas denitrificans cobA are sufficient for the synthesis of cobyric acid in an E. coli cysG deletion strain. Evidence is also presented to suggest that a gene within the S. typhimurium CobI operon might act as a chelatase that, at low levels of cobalt, is able to aid in the synthesis of sirohaem.

scholarly journals New active site oriented glyoxyl-agarose derivatives of Escherichia coli penicillin G acylase

2007 ◽  
Vol 7 (1) ◽  
pp. 54 ◽  
Author(s):  
Davide A Cecchini ◽  
Immacolata Serra ◽  
Daniela Ubiali ◽  
Marco Terreni ◽  
Alessandra M Albertini
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Penicillin G Acylase ◽  
Penicillin G ◽  
Derivatives Of

scholarly journals Serendipitous crystallization and structure determination of cyanase (CynS) fromSerratia proteamaculans

2015 ◽  
Vol 71 (4) ◽  
pp. 471-476 ◽  
Author(s):  
Agata Butryn ◽  
Gabriele Stoehr ◽  
Christian Linke-Winnebeck ◽  
Karl-Peter Hopfner
Keyword(s):  
Escherichia Coli ◽  
Carbon Dioxide ◽  
Structure Determination ◽  
Active Site ◽  
Mass Spectrometric ◽  
Mass Spectrometric Analysis ◽  
Spectrometric Analysis ◽  
Conserved Residues ◽  
Serratia Proteamaculans

Cyanate hydratase (CynS) catalyzes the decomposition of cyanate and bicarbonate into ammonia and carbon dioxide. Here, the serendipitous crystallization of CynS fromSerratia proteamaculans(SpCynS) is reported. SpCynS was crystallized as an impurity and its identity was determined using mass-spectrometric analysis. The crystals belonged to space groupP1 and diffracted to 2.1 Å resolution. The overall structure of SpCynS is very similar to a previously determined structure of CynS fromEscherichia coli. Density for a ligand bound to the SpCynS active site was observed, but could not be unambiguously identified. Additionally, glycerol molecules bound at the entry to the active site of the enzyme indicate conserved residues that might be important for the trafficking of substrates and products.

scholarly journals Structure-function characterization of the conserved regulatory mechanism of the Escherichia coli M48 metalloprotease BepA

2020 ◽  
Author(s):  
Jack A Bryant ◽  
Ian T Cadby ◽  
Zhi-Soon Chong ◽  
Yanina R Sevastsyanovich ◽  
Faye C Morris ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Regulatory Mechanism ◽  
Targeted Mutagenesis ◽  
System Component ◽  
First Line ◽  
Conserved Residues ◽  
Bam Complex ◽  
Function Characterization

The asymmetric Gram-negative outer membrane (OM) is the first line of defence for bacteria against environmental insults and attack by antimicrobials. The key component of the OM is lipopolysaccharide, which is transported to the surface by the essential lipopolysaccharide transport (Lpt) system. Correct folding of the Lpt system component LptD is regulated by a periplasmic metalloprotease, BepA. Here we present the crystal structure of BepA from Escherichia coli, solved to a resolution of 2.18 Å, in which the M48 protease active site is occluded by an active site plug. Informed by our structure, we demonstrate that free movement of the active site plug is essential for BepA function, suggesting that the protein is auto-regulated by the active site plug, which is conserved throughout the M48 metalloprotease family. Targeted mutagenesis of conserved residues reveals that the negative pocket and the TPR cavity are required for function and degradation of the BAM complex component BamA under conditions of stress. Lastly, we show that loss of BepA causes disruption of OM lipid asymmetry, leading to surface exposed phospholipid.

scholarly journals Harnessing natural diversity to identify key amino acid residues in prolidase

2018 ◽  
Author(s):  
Hanna Marie Schilbert ◽  
Vanessa Pellegrinelli ◽  
Sergio Rodriguez-Cuenca ◽  
Antonio Vidal-Puig ◽  
Boas Pucker
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
3D Structure ◽  
Single Amino Acid ◽  
Nucleotide Polymorphisms ◽  
Prolidase Deficiency ◽  
Pathogenic Potential ◽  
Conserved Residues ◽  
Amino Acid Mutations ◽  
Living Organisms

Prolidase (PEPD) catalyses the cleavage of dipeptides with high affinity for proline at the C-terminal end. This function is required in almost all living organisms. In order to detect strongly conserved residues in PEPD, we analysed PEPD orthologous sequences identified in data sets of animals, plants, fungi, archaea, and bacteria. Due to conservation over very long evolutionary time, conserved residues are likely to be of functional relevance. Single amino acid mutations inPEPDcause a disorder called prolidase deficiency and are associated with various cancer types. We provide new insights into 15 additional residues with putative roles in prolidase deficiency and cancer. Moreover, our results confirm previous reports identifying five residues involved in the binding of metal cofactors as highly conserved and enable the classification of several non-synonymous single nucleotide polymorphisms as likely pathogenic and seven as putative polymorphisms. Moreover, more than 50 novel conserved residues across species were identified. Conservation degree per residue across the animal kingdom were mapped to the human PEPD 3D structure revealing the strongest conservation close to the active site accompanied with a higher functional implication and pathogenic potential, validating the importance of a characteristic active site fold for prolidase identity.

scholarly journals Structural Insights into Catalytic Relevances of Substrate Poses in ACC-1

2019 ◽  
Vol 63 (11) ◽  
Author(s):  
Da-Woon Bae ◽  
Ye-Eun Jung ◽  
Young Jun An ◽  
Jung-Hyun Na ◽  
Sun-Shin Cha
Keyword(s):  
Escherichia Coli ◽  
Klebsiella Pneumoniae ◽  
Transition State ◽  
Proteus Mirabilis ◽  
Active Site ◽  
Nucleophilic Attack ◽  
Content Type ◽  
Structural Alterations ◽  
Covalently Bonded ◽  
Structural Insights

ABSTRACT ACC-1 is a plasmid-encoded class C β-lactamase identified in clinical isolates of Klebsiella pneumoniae, Proteus mirabilis, Salmonella enterica, and Escherichia coli. ACC-1-producing bacteria are susceptible to cefoxitin, whereas they are resistant to oxyimino cephalosporins. Here, we depict crystal structures of apo ACC-1, adenylylated ACC-1, and acylated ACC-1 complexed with cefotaxime and cefoxitin. ACC-1 has noteworthy structural alterations in the R2 loop, the Ω loop, and the Phe119 loop located along the active-site rim. The adenylate covalently bonded to the nucleophilic serine reveals a tetrahedral phosphorus mimicking the deacylation transition state. Cefotaxime in ACC-1 has a proper conformation for the substrate-assisted catalysis in that its C-4 carboxylate and N-5 nitrogen are adequately located to facilitate the deacylation reaction. In contrast, cefoxitin in ACC-1 has a distinct conformation, in which those functional groups cannot contribute to catalysis. Furthermore, the orientation of the deacylating water relative to the acyl carbonyl group in ACC-1 is unfavorable for nucleophilic attack.

Structures of c-di-GMP/cGAMP degrading phosphodiesterase VcEAL: identification of a novel conformational switch and its implication

2019 ◽  
Vol 476 (21) ◽  
pp. 3333-3353 ◽  
Author(s):  
Malti Yadav ◽  
Kamalendu Pal ◽  
Udayaditya Sen
Keyword(s):  
Active Site ◽  
Metal Binding ◽  
Metal Ion ◽  
Triosephosphate Isomerase ◽  
Catalytic Mechanism ◽  
Degradation Mechanism ◽  
Structural Basis ◽  
Conserved Residues ◽  
Phosphodiester Bond ◽  
Cyclic Dinucleotides

Cyclic dinucleotides (CDNs) have emerged as the central molecules that aid bacteria to adapt and thrive in changing environmental conditions. Therefore, tight regulation of intracellular CDN concentration by counteracting the action of dinucleotide cyclases and phosphodiesterases (PDEs) is critical. Here, we demonstrate that a putative stand-alone EAL domain PDE from Vibrio cholerae (VcEAL) is capable to degrade both the second messenger c-di-GMP and hybrid 3′3′-cyclic GMP–AMP (cGAMP). To unveil their degradation mechanism, we have determined high-resolution crystal structures of VcEAL with Ca2+, c-di-GMP-Ca2+, 5′-pGpG-Ca2+ and cGAMP-Ca2+, the latter provides the first structural basis of cGAMP hydrolysis. Structural studies reveal a typical triosephosphate isomerase barrel-fold with substrate c-di-GMP/cGAMP bound in an extended conformation. Highly conserved residues specifically bind the guanine base of c-di-GMP/cGAMP in the G2 site while the semi-conserved nature of residues at the G1 site could act as a specificity determinant. Two metal ions, co-ordinated with six stubbornly conserved residues and two non-bridging scissile phosphate oxygens of c-di-GMP/cGAMP, activate a water molecule for an in-line attack on the phosphodiester bond, supporting two-metal ion-based catalytic mechanism. PDE activity and biofilm assays of several prudently designed mutants collectively demonstrate that VcEAL active site is charge and size optimized. Intriguingly, in VcEAL-5′-pGpG-Ca2+ structure, β5–α5 loop adopts a novel conformation that along with conserved E131 creates a new metal-binding site. This novel conformation along with several subtle changes in the active site designate VcEAL-5′-pGpG-Ca2+ structure quite different from other 5′-pGpG bound structures reported earlier.

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