β-Lactones as Privileged Structures for the Active-Site Labeling of Versatile Bacterial Enzyme Classes

2008 ◽  
Vol 47 (24) ◽  
pp. 4600-4603 ◽  
Author(s):  
Thomas Böttcher ◽  
Stephan A. Sieber
Keyword(s):  
Active Site ◽  
Bacterial Enzyme ◽  
Privileged Structures

The crystal structure of Proteus vulgaris tryptophan indole-lyase complexed with oxindolyl-L-alanine: implications for the reaction mechanism

2018 ◽  
Vol 74 (8) ◽  
pp. 748-759
Author(s):  
Robert S. Phillips ◽  
Adriaan A. Buisman ◽  
Sarah Choi ◽  
Anusha Hussaini ◽  
Zachary A. Wood
Keyword(s):  
Crystal Structure ◽  
Hydrogen Bond ◽  
Aromatic Ring ◽  
Active Site ◽  
Enzymatic Reaction ◽  
Proteus Vulgaris ◽  
Bacterial Enzyme ◽  
Small Domain ◽  
Out Of Plane ◽  
Reversible Formation

Tryptophan indole-lyase (TIL) is a bacterial enzyme which catalyzes the reversible formation of indole and ammonium pyruvate from L-tryptophan. Oxindolyl-L-alanine (OIA) is an inhibitor of TIL, with a K i value of about 5 µM. The crystal structure of the complex of Proteus vulgaris TIL with OIA has now been determined at 2.1 Å resolution. The ligand forms a closed quinonoid complex with the pyridoxal 5′-phosphate (PLP) cofactor. The small domain rotates about 10° to close the active site, bringing His458 into position to donate a hydrogen bond to Asp133, which also accepts a hydrogen bond from the heterocyclic NH of the inhibitor. This brings Phe37 and Phe459 into van der Waals contact with the aromatic ring of OIA. Mutation of the homologous Phe464 in Escherichia coli TIL to Ala results in a 500-fold decrease in k cat/K m for L-tryptophan, with less effect on the reaction of other nonphysiological β-elimination substrates. Stopped-flow kinetic experiments of F464A TIL show that the mutation has no effect on the formation of quinonoid intermediates. An aminoacrylate intermediate is observed in the reaction of F464A TIL with S-ethyl-L-cysteine and benzimidazole. A model of the L-tryptophan quinonoid complex with PLP in the active site of P. vulgaris TIL shows that there would be a severe clash of Phe459 (∼1.5 Å apart) and Phe37 (∼2 Å apart) with the benzene ring of the substrate. It is proposed that this creates distortion of the substrate aromatic ring out of plane and moves the substrate upwards on the reaction coordinate towards the transition state, thus reducing the activation energy and accelerating the enzymatic reaction.

scholarly journals Crystal Structure ofStreptococcus pneumoniae N-Acetylglucosamine-1-phosphate Uridyltransferase Bound to Acetyl-coenzyme A Reveals a Novel Active Site Architecture

2000 ◽  
Vol 276 (15) ◽  
pp. 11844-11851 ◽  
Author(s):  
Gerlind Sulzenbacher ◽  
Laurent Gal ◽  
Caroline Peneff ◽  
Florence Fassy ◽  
Yves Bourne
Keyword(s):  
Active Site ◽  
Coenzyme A ◽  
Resistance Mechanisms ◽  
Terminal Region ◽  
Glycopeptide Antibiotics ◽  
Acetyl Coenzyme A ◽  
Acetyl Coenzyme ◽  
Left Handed ◽  
Bacterial Enzyme ◽  
Parallel Fashion

The bifunctional bacterial enzymeN-acetyl-glucosamine-1-phosphate uridyltransferase (GlmU) catalyzes the two-step formation of UDP-GlcNAc, a fundamental precursor in bacterial cell wall biosynthesis. With the emergence of new resistance mechanisms against β-lactam and glycopeptide antibiotics, the biosynthetic pathway of UDP-GlcNAc represents an attractive target for drug design of new antibacterial agents. The crystal structures ofStreptococcus pneumoniaeGlmU in unbound form, in complex with acetyl-coenzyme A (AcCoA) and in complex with both AcCoA and the end product UDP-GlcNAc, have been determined and refined to 2.3, 2.5, and 1.75 Å, respectively. TheS. pneumoniaeGlmU molecule is organized in two separate domains connectedviaa long α-helical linker and associates as a trimer, with the 50-Å-long left-handed β-helix (LβH) C-terminal domains packed against each other in a parallel fashion and the C-terminal region extended far away from the LβH core and exchanged with the β-helix from a neighboring subunit in the trimer. AcCoA binding induces the formation of a long and narrow tunnel, enclosed between two adjacent LβH domains and the interchanged C-terminal region of the third subunit, giving rise to an original active site architecture at the junction of three subunits.

scholarly journals Structure ofEscherichia colitryptophanase purified from an alkaline-stressed bacterial culture

2015 ◽  
Vol 71 (11) ◽  
pp. 1378-1383
Author(s):  
Stephane Rety ◽  
Patrick Deschamps ◽  
Nicolas Leulliot
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Active Site ◽  
Bacterial Culture ◽  
Bacterial Survival ◽  
Space Group ◽  
Bacterial Enzyme

Tryptophanase is a bacterial enzyme involved in the degradation of tryptophan to indole, pyruvate and ammonia, which are compounds that are essential for bacterial survival. Tryptophanase is often overexpressed in stressed cultures. Large amounts of endogenous tryptophanase were purified fromEscherichia coliBL21 strain overexpressing another recombinant protein. Tryptophanase was crystallized in space groupP6522 in the apo form without pyridoxal 5′-phosphate bound in the active site.

scholarly journals Organophosphorus acid anhydrolase fromAlteromonas macleodii: structural study and functional relationship to prolidases

2013 ◽  
Vol 69 (4) ◽  
pp. 346-354 ◽  
Author(s):  
Andrea Štěpánková ◽  
Jarmila Dušková ◽  
Tereza Skálová ◽  
Jindřich Hašek ◽  
Tomáš Koval' ◽  
...  
Keyword(s):  
Active Site ◽  
Three Dimensional ◽  
Size Exclusion ◽  
Dimensional Structure ◽  
Cell Parameters ◽  
Bacterial Enzyme ◽  
Organophosphorus Acid Anhydrolase ◽  
Full Structure ◽  
Exclusion Chromatography ◽  
Pita Bread

The bacterial enzyme organophosphorus acid anhydrolase (OPAA) is able to catalyze the hydrolysis of both proline dipeptides (Xaa-Pro) and several types of organophosphate (OP) compounds. The full three-dimensional structure of the manganese-dependent OPAA enzyme is presented for the first time. This enzyme, which was originally isolated from the marine bacteriumAlteromonas macleodii, was prepared recombinantly inEscherichia coli. The crystal structure was determined at 1.8 Å resolution in space groupC2, with unit-cell parametersa= 133.8,b= 49.2,c= 97.3 Å, β = 125.0°. The enzyme forms dimers and their existence in solution was confirmed by dynamic light scattering and size-exclusion chromatography. The enzyme shares the pita-bread fold of its C-terminal domain with related prolidases. The binuclear manganese centre is located in the active site within the pita-bread domain. Moreover, an Ni2+ion from purification was localized according to anomalous signal. This study presents the full structure of this enzyme with complete surroundings of the active site and provides a critical analysis of its relationship to prolidases.

scholarly journals Indoline-6-Sulfonamide Inhibitors of the Bacterial Enzyme DapE

Antibiotics ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 595
Author(s):  
Cory T. Reidl ◽  
Tahirah K. Heath ◽  
Iman Darwish ◽  
Rachel M. Torrez ◽  
Maxwell Moore ◽  
...  
Keyword(s):  
Molecular Docking ◽  
High Throughput ◽  
Mechanism Of Action ◽  
Active Site ◽  
Diaminopimelic Acid ◽  
Zinc Binding ◽  
High Throughput Screen ◽  
Inhibitory Potency ◽  
Bacterial Enzyme ◽  
Site Binding

Inhibitors of the bacterial enzyme dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE; EC 3.5.1.18) hold promise as antibiotics with a new mechanism of action. Herein we describe the discovery of a new series of indoline sulfonamide DapE inhibitors from a high-throughput screen and the synthesis of a series of analogs. Inhibitory potency was measured by a ninhydrin-based DapE assay recently developed by our group. Molecular docking experiments suggest active site binding with the sulfonamide acting as a zinc-binding group (ZBG).

Two active site arginines are critical determinants of substrate binding and catalysis in MenD: a thiamine-dependent enzyme in menaquinone biosynthesis

2018 ◽  
Vol 475 (22) ◽  
pp. 3651-3667 ◽  
Author(s):  
Mingming Qin ◽  
Haigang Song ◽  
Xin Dai ◽  
Yaozong Chen ◽  
Zhihong Guo
Keyword(s):  
Hydrogen Bonding ◽  
Active Site ◽  
Pathogenic Bacteria ◽  
Site Directed Mutagenesis ◽  
Stetter Reaction ◽  
Enzyme Substrate ◽  
Bacterial Enzyme ◽  
Active Intermediate ◽  
Arginine Residues ◽  
Ionic Hydrogen Bonding

The bacterial enzyme MenD, or 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate (SEPHCHC) synthase, catalyzes an essential Stetter reaction in menaquinone (vitamin K2) biosynthesis via thiamine diphosphate (ThDP)-bound tetrahedral post-decarboxylation intermediates. The detailed mechanism of this intermediate chemistry, however, is still poorly understood, but of significant interest given that menaquinone is an essential electron transporter in many pathogenic bacteria. Here, we used site-directed mutagenesis, enzyme kinetic assays, and protein crystallography to reveal an active–inactive intermediate equilibrium in MenD catalysis and its modulation by two conserved active site arginine residues. We observed that these conserved residues play a key role in shifting the equilibrium to the active intermediate by orienting the C2-succinyl group of the intermediates through strong ionic hydrogen bonding. We found that when this interaction is moderately weakened by amino acid substitutions, the resulting proteins are catalytically competent with the C2-succinyl group taking either the active or the inactive orientation in the post-decarboxylation intermediate. When this hydrogen-bonding interaction was strongly weakened, the succinyl group was re-oriented by 180° relative to the native intermediate, resulting in the reversal of the stereochemistry at the reaction center that disabled catalysis. Interestingly, this inactive intermediate was formed with a distinct kinetic behavior, likely as a result of a non-native mode of enzyme–substrate interaction. The mechanistic insights gained from these findings improve our understanding of the new ThDP-dependent catalysis. More importantly, the non-native-binding site of the inactive MenD intermediate uncovered here provides a new target for the development of antibiotics.

scholarly journals An amino acid sequence in the active site of lipoamide dehydrogenase from pig heart

1974 ◽  
Vol 137 (3) ◽  
pp. 505-512 ◽  
Author(s):  
Joseph P. Brown ◽  
Richard N. Perham
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Active Site ◽  
Amino Acid Sequences ◽  
Disulphide Bridge ◽  
Similar Sequence ◽  
Bacterial Enzyme ◽  
Oxoglutarate Dehydrogenase ◽  
Lipoamide Dehydrogenase ◽  
Polypeptide Chains

1. The two cysteine residues forming the disulphide bridge that comprises part of the active site of lipoamide dehydrogenase from pig heart were specifically labelled with iodo[2-14C]acetic acid. 2. A tryptic peptide containing these carboxymethylcysteine residues was isolated from digests of reduced and S-carboxymethylated lipoamide dehydrogenase and its amino acid sequence of 23 residues was determined. 3. The sequence is highly homologous with a similar sequence containing the active-site disulphide bridge of lipoamide dehydrogenase derived from the 2-oxoglutarate dehydrogenase complex of Escherichia coli (Crookes strain) and it is probable that, as in the bacterial enzyme, the disulphide bridge forms an intrachain loop containing six residues. The results indicate that the bacterial and mammalian proteins have a common genetic origin. 4. Amino acid sequences containing six other unique carboxymethylcysteine residues were also partly determined. 5. The analysis of the primary structure thus far is consistent with the view that the enzyme (mol.wt. approx. 110000) is composed of two identical polypeptide chains.

Mapping the Active Site of the Bacterial Enzyme LpxC Using Novel Carbohydrate‐Based Hydroxamic Acid Inhibitors*

2005 ◽  
Vol 24 (4-6) ◽  
pp. 583-609 ◽  
Author(s):  
Xuechen Li ◽  
Amanda McClerren ◽  
Christian Raetz ◽  
Ole Hindsgaul
Keyword(s):  
Active Site ◽  
Hydroxamic Acid ◽  
Bacterial Enzyme

scholarly journals Irreversible inhibition of Δ5-3-oxosteroid isomerase by 2-substituted progesterones

1985 ◽  
Vol 226 (2) ◽  
pp. 469-476 ◽  
Author(s):  
T M Penning
Keyword(s):  
Active Site ◽  
Covalent Bond ◽  
Competitive Inhibitor ◽  
Enzyme Inactivation ◽  
Nucleophilic Attack ◽  
Dependent Manner ◽  
Compound I ◽  
Irreversible Inhibitors ◽  
Bacterial Enzyme ◽  
Oxosteroid Isomerase

2 alpha-Cyanoprogesterone (I) and 2-hydroxymethyleneprogesterone (II) were synthesized and screened as irreversible active-site-directed inhibitors of the delta 5-3-oxosteroid isomerase (EC 5.3.3.1) from Pseudomonas testosteroni. Both compounds were found to inhibit the purified bacterial enzyme in a time-dependent manner. In either case the inactivated enzyme could be dialysed without return of activity, indicating that a stable covalent bond had formed between the inhibitor and the enzyme. Inactivation mediated by compounds (I) and (II) followed pseudo-first-order kinetics, and at higher inhibitor concentrations saturation was observed. The competitive inhibitor 17 beta-oestradiol offered protection against the inactivation mediated by both compounds, and initial-rate studies indicated that compounds (I) and (II) can also act as competitive inhibitors yielding Ki values identical with those generated during inactivation experiments. 2 alpha-Cyanoprogesterone (I) and 2-hydroxymethyleneprogesterone (II) thus appear to be active-site-directed. To compare the reactivity of these 2-substituted progesterones with other irreversible inhibitors of the isomerase, 3 beta-spiro-oxiranyl-5 alpha-pregnan-20 beta-ol (III) was synthesized as the C21 analogue of 3 beta-spiro-oxiranyl-5 alpha-androstan-17 beta-ol, which is a potent inactivator of the isomerase [Pollack, Kayser & Bevins (1979) Biochem. Biophys. Res. Commun. 91, 783-790]. Comparison of the bimolecular rate constants for inactivation (k+3/Ki) mediated by compounds (I)-(III) indicated the following order of reactivity: (III) greater than (II) greater than (I). 2-Mercaptoethanol offers complete protection against the inactivation of the isomerase mediated by 2 alpha-cyanoprogesterone (I). Under the conditions of inactivation compound (I) appears to be completely stable, and no evidence could be obtained for enolate ion formation in the presence or absence of enzyme. It is suggested that cyanoprogesterone inactivates the isomerase after direct nucleophilic attack at the electropositive 2-position, and that tautomerization plays no role in the inactivation event. By contrast, 2-mercaptoethanol offers no protection against the inactivation mediated by 2-hydroxymethyleneprogesterone, and under the conditions of inactivation this compound appears to exist in the semi-enolized form.

Atomic resolution studies of haloalkane dehalogenases DhaA04, DhaA14 and DhaA15 with engineered access tunnels

2010 ◽  
Vol 66 (9) ◽  
pp. 962-969 ◽  
Author(s):  
A. Stsiapanava ◽  
J. Dohnalek ◽  
J. A. Gavira ◽  
M. Kuty ◽  
T. Koudelakova ◽  
...  
Keyword(s):  
Active Site ◽  
Ligand Exchange ◽  
Hydrolytic Degradation ◽  
Haloalkane Dehalogenase ◽  
Orthorhombic Space Group ◽  
Space Group ◽  
Diffusion Technique ◽  
Bacterial Enzyme ◽  
Functional Relevance ◽  
Protein Variants

The haloalkane dehalogenase DhaA fromRhodococcus rhodochrousNCIMB 13064 is a bacterial enzyme that shows catalytic activity for the hydrolytic degradation of the highly toxic industrial pollutant 1,2,3-trichloropropane (TCP). Mutagenesis focused on the access tunnels of DhaA produced protein variants with significantly improved activity towards TCP. Three mutants of DhaA named DhaA04 (C176Y), DhaA14 (I135F) and DhaA15 (C176Y + I135F) were constructed in order to study the functional relevance of the tunnels connecting the buried active site of the protein with the surrounding solvent. All three protein variants were crystallized using the sitting-drop vapour-diffusion technique. The crystals of DhaA04 belonged to the orthorhombic space groupP212121, while the crystals of DhaA14 and DhaA15 had triclinic symmetry in space groupP1. The crystal structures of DhaA04, DhaA14 and DhaA15 with ligands present in the active site were solved and refined using diffraction data to 1.23, 0.95 and 1.22 Å, resolution, respectively. Structural comparisons of the wild type and the three mutants suggest that the tunnels play a key role in the processes of ligand exchange between the buried active site and the surrounding solvent.

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