scholarly journals Bicyclic Boronates as Potent Inhibitors of AmpC, the Class C β-Lactamase from Escherichia coli

Biomolecules ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 899 ◽  
Author(s):  
Pauline A. Lang ◽  
Anete Parkova ◽  
Thomas M. Leissing ◽  
Karina Calvopiña ◽  
Ricky Cain ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
High Energy ◽  
Structural Basis ◽  
Side Chain ◽  
Class A ◽  
Dual Action ◽  
Class C ◽  
Tract Infections ◽  
Site Binding

Resistance to β-lactam antibacterials, importantly via production of β-lactamases, threatens their widespread use. Bicyclic boronates show promise as clinically useful, dual-action inhibitors of both serine- (SBL) and metallo- (MBL) β-lactamases. In combination with cefepime, the bicyclic boronate taniborbactam is in phase 3 clinical trials for treatment of complicated urinary tract infections. We report kinetic and crystallographic studies on the inhibition of AmpC, the class C β-lactamase from Escherichia coli, by bicyclic boronates, including taniborbactam, with different C-3 side chains. The combined studies reveal that an acylamino side chain is not essential for potent AmpC inhibition by active site binding bicyclic boronates. The tricyclic form of taniborbactam was observed bound to the surface of crystalline AmpC, but not at the active site, where the bicyclic form was observed. Structural comparisons reveal insights into why active site binding of a tricyclic form has been observed with the NDM-1 MBL, but not with other studied β-lactamases. Together with reported studies on the structural basis of inhibition of class A, B and D β-lactamases, our data support the proposal that bicyclic boronates are broad-spectrum β-lactamase inhibitors that work by mimicking a high energy ‘tetrahedral’ intermediate. These results suggest further SAR guided development could improve the breadth of clinically useful β-lactamase inhibition.

scholarly journals Structural Basis for Imipenem Inhibition of Class C β-Lactamases

2002 ◽  
Vol 46 (12) ◽  
pp. 3978-3980 ◽  
Author(s):  
Beth M. Beadle ◽  
Brian K. Shoichet
Keyword(s):  
Crystal Structure ◽  
Carbonyl Oxygen ◽  
Structural Basis ◽  
Enzyme Complex ◽  
X Ray ◽  
Class A ◽  
Lactam Carbonyl ◽  
Class C ◽  
Expected Position

ABSTRACT To determine how imipenem inhibits the class C β-lactamase AmpC, the X-ray crystal structure of the acyl-enzyme complex was determined to a resolution of 1.80 Å. In the complex, the lactam carbonyl oxygen of imipenem has flipped by approximately 180° compared to its expected position; the electrophilic acyl center is thus displaced from the point of hydrolytic attack. This conformation resembles that of imipenem bound to the class A enzyme TEM-1 but is different from that of moxalactam bound to AmpC.

scholarly journals Crystal structures of two monomeric triosephosphate isomerase variants identifiedviaa directed-evolution protocol selecting forL-arabinose isomerase activity

2016 ◽  
Vol 72 (6) ◽  
pp. 490-499
Author(s):  
Mirja Krause ◽  
Tiila-Riikka Kiema ◽  
Peter Neubauer ◽  
Rik K. Wierenga
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Directed Evolution ◽  
Crystal Structures ◽  
Active Site ◽  
Triosephosphate Isomerase ◽  
Point Mutations ◽  
Van Der Waals Interactions ◽  
Side Chain ◽  
Arabinose Isomerase

The crystal structures are described of two variants of A-TIM: Ma18 (2.7 Å resolution) and Ma21 (1.55 Å resolution). A-TIM is a monomeric loop-deletion variant of triosephosphate isomerase (TIM) which has lost the TIM catalytic properties. Ma18 and Ma21 were identified after extensive directed-evolution selection experiments using anEscherichia coliL-arabinose isomerase knockout strain expressing a randomly mutated A-TIM gene. These variants facilitate better growth of theEscherichia coliselection strain in medium supplemented with 40 mML-arabinose. Ma18 and Ma21 differ from A-TIM by four and one point mutations, respectively. Ma18 and Ma21 are more stable proteins than A-TIM, as judged from CD melting experiments. Like A-TIM, both proteins are monomeric in solution. In the Ma18 crystal structure loop 6 is open and in the Ma21 crystal structure loop 6 is closed, being stabilized by a bound glycolate molecule. The crystal structures show only small differences in the active site compared with A-TIM. In the case of Ma21 it is observed that the point mutation (Q65L) contributes to small structural rearrangements near Asn11 of loop 1, which correlate with different ligand-binding properties such as a loss of citrate binding in the active site. The Ma21 structure also shows that its Leu65 side chain is involved in van der Waals interactions with neighbouring hydrophobic side-chain moieties, correlating with its increased stability. The experimental data suggest that the increased stability and solubility properties of Ma21 and Ma18 compared with A-TIM cause better growth of the selection strain when coexpressing Ma21 and Ma18 instead of A-TIM.

scholarly journals The aminoacylation of transfer ribonucleic acid. Recognition of methionine by Escherichia coli methionyl-transfer ribonucleic acid synthetase

1977 ◽  
Vol 165 (2) ◽  
pp. 367-373 ◽  
Author(s):  
J M Old ◽  
D S Jones
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Ribonucleic Acid ◽  
Chemical Nature ◽  
Trna Synthetase ◽  
Side Chain ◽  
Amino Acid Side Chain ◽  
Sulphur Atom ◽  
Recognition Mechanism ◽  
Methionyl Trna Synthetase

The mechanism of the recognition of methionine by Escherichia coli methionyl-tRNA synthetase was examined by a kinetic study of the recognition of methionine analogues in the ATP-PPi exchange reaction and the tRNA-aminoacylation reaction. The results show that the recognition mechanism consists of three parts: (1) the recognition of the size, shape and chemical nature of the amino acid side chain at the methionine-binding stage of the reaction; (2) the recognition of the length of the side chain at the stage of aminoacyl-adenylate complex-formation; (3) the recognition of the sulphur atom in the side chain at the stage of methionyl-tRNA formation. It is proposed that the sulphur atom interacts with the enzyme to induce a conformational change. A model of the active site incorporating the mechanism of methionine recognition is presented.

scholarly journals High-Resolution Crystal Structure of the Subclass B3 Metallo-β-Lactamase BJP-1: Rational Basis for Substrate Specificity and Interaction with Sulfonamides

2010 ◽  
Vol 54 (10) ◽  
pp. 4343-4351 ◽  
Author(s):  
Jean-Denis Docquier ◽  
Manuela Benvenuti ◽  
Vito Calderone ◽  
Magdalena Stoczko ◽  
Nicola Menciassi ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Bradyrhizobium Japonicum ◽  
Active Sites ◽  
Structural Diversity ◽  
Binding Pocket ◽  
Structural Basis ◽  
Side Chain ◽  
Functional Heterogeneity ◽  
Hydrophobic Binding

ABSTRACT Metallo-β-lactamases (MBLs) are important enzymatic factors in resistance to β-lactam antibiotics that show important structural and functional heterogeneity. BJP-1 is a subclass B3 MBL determinant produced by Bradyrhizobium japonicum that exhibits interesting properties. BJP-1, like CAU-1 of Caulobacter vibrioides, overall poorly recognizes β-lactam substrates and shows an unusual substrate profile compared to other MBLs. In order to understand the structural basis of these properties, the crystal structure of BJP-1 was obtained at 1.4-Å resolution. This revealed significant differences in the conformation and locations of the active-site loops, determining a rather narrow active site and the presence of a unique N-terminal helix bearing Phe-31, whose side chain binds in the active site and represents an obstacle for β-lactam substrate binding. In order to probe the potential of sulfonamides (known to inhibit various zinc-dependent enzymes) to bind in the active sites of MBLs, the structure of BJP-1 in complex with 4-nitrobenzenesulfonamide was also obtained (at 1.33-Å resolution), thereby revealing the mode of interaction of these molecules in MBLs. Interestingly, sulfonamide binding resulted in the displacement of the side chain of Phe-31 from its hydrophobic binding pocket, where the benzene ring of the molecule is now found. These data further highlight the structural diversity shown by MBLs but also provide interesting insights in the structure-function relationships of these enzymes. More importantly, we provided the first structural observation of MBL interaction with sulfonamides, which might represent an interesting scaffold for the design of MBL inhibitors.

scholarly journals Modified Penicillin Molecule with Carbapenem-Like Stereochemistry Specifically Inhibits Class C β-Lactamases

2017 ◽  
Vol 61 (12) ◽  
Author(s):  
Xuehua Pan ◽  
Yunjiao He ◽  
Tianfeng Chen ◽  
Kin-Fai Chan ◽  
Yanxiang Zhao
Keyword(s):  
Active Site ◽  
Binding Mode ◽  
Carboxyl Group ◽  
Hydrogen Atoms ◽  
Chemical Modifications ◽  
Inhibitory Potency ◽  
Current Collection ◽  
Class A ◽  
Lactam Ring ◽  
Class C

ABSTRACT Bacterial β-lactamases readily inactivate most penicillins and cephalosporins by hydrolyzing and “opening” their signature β-lactam ring. In contrast, carbapenems resist hydrolysis by many serine-based class A, C, and D β-lactamases due to their unique stereochemical features. To improve the resistance profile of penicillins, we synthesized a modified penicillin molecule, MPC-1, by “grafting” carbapenem-like stereochemistry onto the penicillin core. Chemical modifications include the trans conformation of hydrogen atoms at C-5 and C-6 instead of cis, and a 6-α hydroxyethyl moiety to replace the original 6-β aminoacyl group. MPC-1 selectively inhibits class C β-lactamases, such as P99, by forming a nonhydrolyzable acyl adduct, and its inhibitory potency is ∼2 to 5 times higher than that for clinically used β-lactamase inhibitors clavulanate and sulbactam. The crystal structure of MPC-1 forming the acyl adduct with P99 reveals a novel binding mode for MPC-1 that resembles carbapenem bound in the active site of class A β-lactamases. Furthermore, in this novel binding mode, the carboxyl group of MPC-1 blocks the deacylation reaction by occluding the critical catalytic water molecule and renders the acyl adduct nonhydrolyzable. Our results suggest that by incorporating carbapenem-like stereochemistry, the current collection of over 100 penicillins and cephalosporins can be modified into candidate compounds for development of novel β-lactamase inhibitors.

scholarly journals The diversity of the catalytic properties of class A β-lactamases

1990 ◽  
Vol 265 (1) ◽  
pp. 131-146 ◽  
Author(s):  
A Matagne ◽  
A M Misselyn-Bauduin ◽  
B Joris ◽  
T Erpicum ◽  
B Granier ◽  
...  
Keyword(s):  
Catalytic Properties ◽  
Amino Acid Sequences ◽  
Side Chain ◽  
Beta Lactam ◽  
Beta Lactamases ◽  
Class A ◽  
Wide Range ◽  
Class C ◽  
Range Of Variation ◽  
Sequence Similarities

The catalytic properties of four class A beta-lactamases were studied with 24 different substrates. They exhibit a wide range of variation. Similarly, the amino acid sequences are also quite different. However, no relationships were found between the sequence similarities and the substrate profiles. Lags and bursts were observed with various compounds containing a large sterically hindered side chain. As a group, the enzymes could be distinguished from the class C beta-lactamases on the basis of the kappa cat. values for several substrates, particularly oxacillin, cloxacillin and carbenicillin. Surprisingly, that distinction was impossible with the kappa cat./Km values, which represent the rates of acylation of the active-site serine residue by the beta-lactam. For several cephalosporin substrates (e.g. cefuroxime and cefotaxime) class A enzymes consistently exhibited higher kappa cat. values than class C enzymes, thus belying the usual distinction between ‘penicillinases’ and ‘cephalosporinases’. The problem of the repartition of class A beta-lactamases into sub-classes is discussed.

scholarly journals The mutation Lys234His yields a class A β-lactamase with a novel pH-dependence

1991 ◽  
Vol 278 (3) ◽  
pp. 673-678 ◽  
Author(s):  
J Brannigan ◽  
A Matagne ◽  
F Jacob ◽  
C Damblon ◽  
B Joris ◽  
...  
Keyword(s):  
Active Site ◽  
Positive Charge ◽  
Ph Dependence ◽  
Side Chain ◽  
Beta Lactamase ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Beta Lactamases ◽  
Class A ◽  
Transition State Stabilization

The lysine-234 residue is highly conserved in beta-lactamases and in nearly all active-site-serine penicillin-recognizing enzymes. Its replacement by a histidine residue in the Streptomyces albus G class A beta-lactamase yielded an enzyme the pH-dependence of which was characterized by the appearance of a novel pK, which could be attributed to the newly introduced residue. At low pH, the kcat, value for benzylpenicillin was as high as 50% of that of the wild-type enzyme, demonstrating that an efficient active site was maintained. Both kcat. and kcat/Km dramatically decreased above pH 6 but the decrease in kcat./Km could not be attributed to larger Km values. Thus a positive charge on the side chain of residue 234 appears to be more essential for transition-state stabilization than for initial recognition of the substrate ground state.

scholarly journals A Novel Type of AmpC β-Lactamase, ACC-1, Produced by a Klebsiella pneumoniae Strain Causing Nosocomial Pneumonia

1999 ◽  
Vol 43 (8) ◽  
pp. 1924-1931 ◽  
Author(s):  
Adolf Bauernfeind ◽  
Ines Schneider ◽  
Renate Jungwirth ◽  
Hany Sahly ◽  
Uwe Ullmann
Keyword(s):  
Escherichia Coli ◽  
Klebsiella Pneumoniae ◽  
Nosocomial Pneumonia ◽  
Active Site ◽  
Multiple Alignment ◽  
Transmissible Plasmid ◽  
Phenotype Characteristic ◽  
Class 1 ◽  
New Type ◽  
Class C

ABSTRACT A Klebsiella pneumoniae strain resistant to oxyimino cephalosporins was cultured from respiratory secretions of a patient suffering from nosocomial pneumonia in Kiel, Germany, in 1997. The isolate harbors a bla resistance gene located on a transmissible plasmid. An Escherichia coli transconjugant produces a β-lactamase with an isoelectric point of 7.7 and a resistance phenotype characteristic of an AmpC (class 1) β-lactamase except for low MICs of cephamycins. Thebla gene was cloned and sequenced. It encodes a protein of 386 amino acids with the active site serine of the S-X-X-K motif at position 64, as is characteristic for class C β-lactamases. Multiple alignment of the deduced amino acid sequence with 21 other AmpC β-lactamases demonstrates only very distant homology, reaching at maximum 52.3% identity for the chromosomal AmpC β-lactamase ofSerratia marcescens SR50. The β-lactamase ofK. pneumoniae KUS represents a new type of AmpC-class enzyme, for which we propose the designation ACC-1 (Ambler class C-1).

scholarly journals Structural Basis for Broad Substrate Selectivity of Alcohol Dehydrogenase YjgB from Escherichia coli

Molecules ◽  
2020 ◽  
Vol 25 (10) ◽  
pp. 2404
Author(s):  
Giang Thu Nguyen ◽  
Yeon-Gil Kim ◽  
Jae-Woo Ahn ◽  
Jeong Ho Chang
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Nicotinamide Adenine Dinucleotide ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Docking Simulation ◽  
Structural Basis ◽  
Aliphatic Chain ◽  
Substrate Selectivity ◽  
Hydrophobic Pocket ◽  
Simulation Data

In metabolic engineering and synthetic biology fields, there have been efforts to produce variable bioalcohol fuels, such as isobutanol and 2-phenylethanol, in order to meet industrial demands. YjgB is an aldehyde dehydrogenase from Escherichia coli that shows nicotinamide adenine dinucleotide phosphate (NADP)-dependent broad selectivity for aldehyde derivatives with an aromatic ring or small aliphatic chain. This could contribute to the design of industrial synthetic pathways. We determined the crystal structures of YjgB for both its apo-form and NADP-complexed form at resolutions of 1.55 and 2.00 Å, respectively, in order to understand the mechanism of broad substrate selectivity. The hydrophobic pocket of the active site and the nicotinamide ring of NADP(H) are both involved in conferring its broad specificity toward aldehyde substrates. In addition, based on docking-simulation data, we inferred that π–π stacking between substrates and aromatic side chains might play a crucial role in recognizing substrates. Our structural analysis of YjgB might provide insights into establishing frameworks to understand its broad substrate specificity and develop engineered enzymes for industrial biofuel synthesis.

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