scholarly journals Structure of the phosphotransferase domain of the bifunctional aminoglycoside-resistance enzyme AAC(6′)-Ie-APH(2′′)-Ia

2014 ◽  
Vol 70 (6) ◽  
pp. 1561-1571 ◽  
Author(s):  
Clyde A. Smith ◽  
Marta Toth ◽  
Monolekha Bhattacharya ◽  
Hilary Frase ◽  
Sergei B. Vakulenko
Keyword(s):  
Active Site ◽  
Domain Architecture ◽  
Aminoglycoside Antibiotics ◽  
Side Chain ◽  
Aminoglycoside Resistance ◽  
Gram Positive Bacteria ◽  
Dual Specificity ◽  
Structural Details ◽  
Aminoglycoside Phosphotransferases ◽  
Almost All

The bifunctional acetyltransferase(6′)-Ie-phosphotransferase(2′′)-Ia [AAC(6′)-Ie-APH(2′′)-Ia] is the most important aminoglycoside-resistance enzyme in Gram-positive bacteria, conferring resistance to almost all known aminoglycoside antibiotics in clinical use. Owing to its importance, this enzyme has been the focus of intensive research since its isolation in the mid-1980s but, despite much effort, structural details of AAC(6′)-Ie-APH(2′′)-Ia have remained elusive. The structure of the Mg2GDP complex of the APH(2′′)-Ia domain of the bifunctional enzyme has now been determined at 2.3 Å resolution. The structure of APH(2′′)-Ia is reminiscent of the structures of other aminoglycoside phosphotransferases, having a two-domain architecture with the nucleotide-binding site located at the junction of the two domains. Unlike the previously characterized APH(2′′)-IIa and APH(2′′)-IVa enzymes, which are capable of utilizing both ATP and GTP as the phosphate donors, APH(2′′)-Ia uses GTP exclusively in the phosphorylation of the aminoglycoside antibiotics, and in this regard closely resembles the GTP-dependent APH(2′′)-IIIa enzyme. In APH(2′′)-Ia this GTP selectivity is governed by the presence of a `gatekeeper' residue, Tyr100, the side chain of which projects into the active site and effectively blocks access to the adenine-binding template. Mutation of this tyrosine residue to a less bulky phenylalanine provides better access for ATP to the NTP-binding template and converts APH(2′′)-Ia into a dual-specificity enzyme.

scholarly journals Chemical modification of serine at the active site of penicillin acylase from Kluyvera citrophila

1991 ◽  
Vol 280 (3) ◽  
pp. 659-662 ◽  
Author(s):  
J Martín ◽  
A Slade ◽  
A Aitken ◽  
R Arche ◽  
R Virden
Keyword(s):  
Active Site ◽  
Tertiary Structure ◽  
Thiol Group ◽  
Iodoacetic Acid ◽  
Penicillin Acylase ◽  
Protein Product ◽  
Side Chain ◽  
Serine Residue ◽  
Conserved Sequence ◽  
Ester Substrate

The site of reaction of penicillin acylase from Kluyvera citrophila with the potent inhibitor phenylmethanesulphonyl fluoride was investigated by incubating the inactivated enzyme with thioacetic acid to convert the side chain of the putative active-site serine residue to that of cysteine. The protein product contained one thiol group, which was reactive towards 2,2′-dipyridyl disulphide and iodoacetic acid. Carboxymethylcysteine was identified as the N-terminal residue of the beta-subunit of the carboxy[3H]methylthiol-protein. No significant changes in tertiary structure were detected in the modified penicillin acylase using near-u.v. c.d. spectroscopy. However, the catalytic activity (kcat) with either an anilide or an ester substrate was decreased in the thiol-protein by a factor of more than 10(4). A comparison of sequences of apparently related acylases shows no other extensive regions of conserved sequence containing an invariant serine residue. The side chain of this residue is proposed as a candidate nucleophile in the formation of an acyl-enzyme during catalysis.

Crystal structure of acetylxylan esterase from Caldanaerobacter subterraneus subsp. tengcongensis

2021 ◽  
Vol 77 (11) ◽  
Author(s):  
Kohei Sasamoto ◽  
Tomoki Himiyama ◽  
Kunihiko Moriyoshi ◽  
Takashi Ohmoto ◽  
Koichi Uegaki ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Cellulose Acetate ◽  
Electron Density ◽  
Active Site ◽  
Catalytic Domain ◽  
Crystal Packing ◽  
Signal Sequence ◽  
Industrial Applications ◽  
Side Chain ◽  
Active Site Conformation

The acetylxylan esterases (AXEs) classified into carbohydrate esterase family 4 (CE4) are metalloenzymes that catalyze the deacetylation of acetylated carbohydrates. AXE from Caldanaerobacter subterraneus subsp. tengcongensis (TTE0866), which belongs to CE4, is composed of three parts: a signal sequence (residues 1–22), an N-terminal region (NTR; residues 23–135) and a catalytic domain (residues 136–324). TTE0866 catalyzes the deacetylation of highly substituted cellulose acetate and is expected to be useful for industrial applications in the reuse of resources. In this study, the crystal structure of TTE0866 (residues 23–324) was successfully determined. The crystal diffracted to 1.9 Å resolution and belonged to space group I212121. The catalytic domain (residues 136–321) exhibited a (β/α)7-barrel topology. However, electron density was not observed for the NTR (residues 23–135). The crystal packing revealed the presence of an intermolecular space without observable electron density, indicating that the NTR occupies this space without a defined conformation or was truncated during the crystallization process. Although the active-site conformation of TTE0866 was found to be highly similar to those of other CE4 enzymes, the orientation of its Trp264 side chain near the active site was clearly distinct. The unique orientation of the Trp264 side chain formed a different-shaped cavity within TTE0866, which may contribute to its reactivity towards highly substituted cellulose acetate.

Conformation–activity relationships and the mechanism of action of penicillin

1988 ◽  
Vol 66 (11) ◽  
pp. 2733-2750 ◽  
Author(s):  
Saul Wolfe ◽  
Kiyull Yang ◽  
Maged Khalil
Keyword(s):  
Conformational Analysis ◽  
Carbonyl Group ◽  
Active Site ◽  
Global Minimum ◽  
Peptide Bond ◽  
Biologically Active ◽  
Penicillin G ◽  
Side Chain ◽  
Geometrical Parameters ◽  
Carboxyl Group

Using the MMPEN parameters of Allinger's MMP2(85) force field, a conformational analysis has been performed on four biologically active penicillins; D-ampicillin, L-α-phenoxyethylpenicillin, penicillin G, and penicillin V, and on five biologically inactive or much less active penicillins: L-ampicillin, D-α-phenoxyethylpenicillin, N-methylpenicillin G, 6α-methylpenicillin G, and bisnorpenicillin G. Antibacterial activity is found to be associated with the existence of a global minimum having a compact structure, whose convex face is accessible to a penicillin binding protein (PBP), with the C3-carboxyl group and the side-chain N-H exposed on this face. Using the MMPEP parameters of MMP2(85), a conformational analysis has been performed on phenylacetyl-D-Ala-D-Ala-O−, a peptide model of the normal substrate of a PBP. Labischinski's global minimum has been reproduced, along with structures that correspond to Tipper and Strominger's proposal that the N4—C7 bond of a penicillin corresponds to the Ala–Ala peptide bond, and to Hasan's proposal that the N4—C5 bond of penicillin corresponds to the peptide bond. For both models, conformations of the peptide related to the pseudoaxial and pseudoequatorial conformations of the thiazolidine ring of penicillin G have been examined. It is concluded that penicillin is not a structural analog of the global minimum of the peptide; however, comparisons based on unbound conformations of PBP substrates are unable to determine which model is more appropriate, or which conformation of penicillin G is the biologically significant one. Using the ECEPP/MMPEP strategy, a model of the active site of a PBP has been obtained, following a search of 200,000 structures of the peptide Ac-NH-Val-Gly-Ser-Val-Thr-Lys-NH-Me. This peptide contains the sequence at the active site of a PBP of Streptomyces R61, for which it is also known that the C3-carboxyl group of penicillin binds to the ε-amino group of lysine, and the β-lactam reacts chemically with the serine OH. The lysine and serine side chains and the C-terminal carbonyl group are found to occupy the concave face of the active site model.A strategy for the docking of penicillins or peptides to this model, with full minimization of the conformational energies of the complexes, has been devised. All active penicillins bind through strong hydrogen bonds to the C3-carboxyl group and the side-chain N-H, and with a four-centered relationship between the O-H of serine and the (O)C-N of the β-lactam ring. The geometrical parameters of this relationship are reminiscent of those found in the gas phase transition state of neutral hydration of a carbonyl group. When the energies of formation and geometries of the pseudoaxial and pseudoequatorial penicillin G complexes are examined, there is now a clear preference for the binding of the pseudoaxial conformation, which is the global minimum of the uncomplexed penicillin in this case. A similar examination of the peptide complexes reveals that only the conformation of the peptide that corresponds to Tipper and Strominger's model, and is based on the pseudoaxial conformation of penicillin G, can form a complex with a geometry and energy comparable to those of a biologically active penicillin.

scholarly journals An Adaptive Geometric Search Algorithm for Macromolecular Scaffold Selection

2017 ◽  
Author(s):  
Tian Jiang ◽  
P. Douglas Renfrew ◽  
Kevin Drew ◽  
Noah Youngs ◽  
Glenn Butterfoss ◽  
...  
Keyword(s):  
Protein Design ◽  
Active Site ◽  
Metal Binding ◽  
Search Algorithm ◽  
Three Dimensional ◽  
Side Chain ◽  
Enzyme Design ◽  
Hydrogen Binding ◽  
Performance Improvements ◽  
Geometric Search

AbstractA wide variety of protein and peptidomimetic design tasks require matching functional three-dimensional motifs to potential oligomeric scaffolds. Enzyme design, for example, aims to graft active-site patterns typically consisting of 3 to 15 residues onto new protein surfaces. Identifying suitable proteins capable of scaffolding such active-site engraftment requires costly searches to identify protein folds that can provide the correct positioning of side chains to host the desired active site. Other examples of biodesign tasks that require simpler fast exact geometric searches of potential side chain positioning include mimicking binding hotspots, design of metal binding clusters and the design of modular hydrogen binding networks for specificity. In these applications the speed and scaling of geometric search limits downstream design to small patterns. Here we present an adaptive algorithm to searching for side chain take-off angles compatible with an arbitrarily specified functional pattern that enjoys substantive performance improvements over previous methods. We demonstrate this method in both genetically encoded (protein) and synthetic (peptidomimetic) design scenarios. Examples of using this method with the Rosetta framework for protein design are provided but our implementation is compatible with multiple protein design frameworks and is freely available as a set of python scripts (https://github.com/JiangTian/adaptive-geometric-search-for-protein-design).

scholarly journals X-ray structure of a putative reaction intermediate of 5-aminolaevulinic acid dehydratase

2003 ◽  
Vol 373 (3) ◽  
pp. 733-738 ◽  
Author(s):  
Peter T. ERSKINE ◽  
Leighton COATES ◽  
Danica BUTLER ◽  
James H. YOUELL ◽  
Amanda A. BRINDLEY ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Mechanism ◽  
Zinc Ion ◽  
Side Chain ◽  
Hydroxide Ion ◽  
X Ray ◽  
Aminolaevulinic Acid ◽  
Acid Dehydratase ◽  
Cyclic Intermediate ◽  
Aminolaevulinic Acid Dehydratase

The X-ray structure of yeast 5-aminolaevulinic acid dehydratase, in which the catalytic site of the enzyme is complexed with a putative cyclic intermediate composed of both substrate moieties, has been solved at 0.16 nm (1.6 Å) resolution. The cyclic intermediate is bound covalently to Lys263 with the amino group of the aminomethyl side chain ligated to the active-site zinc ion in a position normally occupied by a catalytic hydroxide ion. The cyclic intermediate is catalytically competent, as shown by its turnover in the presence of added substrate to form porphobilinogen. The findings, combined with those of previous studies, are consistent with a catalytic mechanism in which the C–C bond linking both substrates in the intermediate is formed before the C–N bond.

scholarly journals Antimicrobial Porous Surfaces Prepared by Breath Figures Approach

Materials ◽  
2018 ◽  
Vol 11 (8) ◽  
pp. 1266 ◽  
Author(s):  
Alexandra Muñoz-Bonilla ◽  
Rocío Cuervo-Rodríguez ◽  
Fátima López-Fabal ◽  
José Gómez-Garcés ◽  
Marta Fernández-García
Keyword(s):  
Block Copolymers ◽  
Side Chain ◽  
Porous Films ◽  
Narrow Pore Size Distribution ◽  
Breath Figures ◽  
Gram Positive Bacteria ◽  
Alkyl Groups ◽  
Porous Surfaces ◽  
Cationic Copolymers ◽  
Ordered Porous

Herein, efficient antimicrobial porous surfaces were prepared by breath figures approach from polymer solutions containing low content of block copolymers with high positive charge density. In brief, those block copolymers, which were used as additives, are composed of a polystyrene segment and a large antimicrobial block bearing flexible side chain with 1,3-thiazolium and 1,2,3-triazolium groups, PS54-b-PTTBM-M44, PS54-b-PTTBM-B44, having different alkyl groups, methyl or butyl, respectively. The antimicrobial block copolymers were blended with commercial polystyrene in very low proportions, from 3 to 9 wt %, and solubilized in THF. From these solutions, ordered porous films functionalized with antimicrobial cationic copolymers were fabricated, and the influence of alkylating agent and the amount of copolymer in the blend was investigated. Narrow pore size distribution was obtained for all the samples with pore diameters between 5 and 11 µm. The size of the pore decreased as the hydrophilicity of the system increased; thus, either as the content of copolymer was augmented in the blend or as the copolymers were quaternized with methyl iodide. The resulting porous polystyrene surfaces functionalized with low content of antimicrobial copolymers exhibited remarkable antibacterial efficiencies against Gram positive bacteria Staphylococcus aureus, and Candida parapsilosis fungi as microbial models.

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