Allosteric communication in class A β-lactamases occurs via cooperative coupling of loop dynamics.
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ABSTRACT:
SUBMITTER: Galdadas I
PROVIDER: S-EPMC8060031 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:A set of 49 high-resolution (<or=2.2 A) structures of the TEM, SHV, and CTX-M class A beta-lactamase families was systematically analyzed to investigate the role of conserved water molecules in the stabilization of the Omega-loop. Overall, 13 water molecules were found to be conserved in at least 45 structures, including two water positions which were found to be conserved in all structures. Of the 13 conserved water molecules, 6 are located at the Omega-loop, forming a dense cluster with hydrogen bonds to residues at the Omega-loop as well as to the rest of the protein. This layer of conserved water molecules is packed between the Omega-loop and the rest of the protein and acts as structural glue, which could reduce the flexibility of the Omega-loop. A correlation between conserved water molecules and conserved protein residues could in general not be detected, with the exception of the conserved water molecules at the Omega-loop. Furthermore, the evolutionary relationship between the three families, derived from the number of conserved water molecules, is similar to the relationship derived from phylogenetic analysis.
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Project description:The expression of β-lactamases is a major mechanism of bacterial resistance to the β-lactam antibiotics. Four molecular classes of β-lactamases have been described (A, B, C and D), however until recently the class D enzymes were thought to exist only in Gram-negative bacteria. In the last few years, class D enzymes have been discovered in several species of Gram-positive microorganisms, such as Bacillus and Clostridia, and an investigation of their kinetic and structural properties has begun in earnest. Interestingly, it was observed that some species of Bacillus produce two distinct class D β-lactamases, one highly active and the other with only basal catalytic activity. Analysis of amino acid sequences of active (BPU-1 from Bacillus pumilus) and inactive (BSU-2 from Bacillus subtilis and BAT-2 from Bacillus atrophaeus) enzymes suggests that presence of three additional amino acid residues in one of the surface loops of inefficient β-lactamases may be responsible for their severely diminished activity. Our structural and docking studies show that the elongated loop of these enzymes severely restricts binding of substrates. Deletion of the three residues from the loops of BSU-2 and BAT-2 β-lactamases relieves the steric hindrance and results in a significant increase in the catalytic activity of the enzymes. These data show that this surface loop plays an important role in modulation of the catalytic activity of Bacillus class D β-lactamases.
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Project description:Class A β-lactamases are known for being able to rapidly gain broad spectrum catalytic efficiency against most β-lactamase inhibitor combinations as a result of elusively minor point mutations. The evolution in class A β-lactamases occurs through optimisation of their dynamic phenotypes at different timescales. At long-timescales, certain conformations are more catalytically permissive than others while at the short timescales, fine-grained optimisation of free energy barriers can improve efficiency in ligand processing by the active site. Free energy barriers, which define all coordinated movements, depend on the flexibility of the secondary structural elements. The most highly conserved residues in class A β-lactamases are hydrophobic nodes that stabilize the core. To assess how the stable hydrophobic core is linked to the structural dynamics of the active site, we carried out adaptively sampled molecular dynamics (MD) simulations in four representative class A β-lactamases (KPC-2, SME-1, TEM-1, and SHV-1). Using Markov State Models (MSM) and unsupervised deep learning, we show that the dynamics of the hydrophobic nodes is used as a metastable relay of kinetic information within the core and is coupled with the catalytically permissive conformation of the active site environment. Our results collectively demonstrate that the class A enzymes described here, share several important dynamic similarities and the hydrophobic nodes comprise of an informative set of dynamic variables in representative class A β-lactamases.
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Project description:OXA beta-lactamases are largely responsible for beta-lactam resistance in Acinetobacter spp. and Pseudomonas aeruginosa, two of the most difficult-to-treat nosocomial pathogens. In general, the beta-lactamase inhibitors used in clinical practice (clavulanic acid, sulbactam, and tazobactam) demonstrate poor activity against class D beta-lactamases. To overcome this challenge, we explored the abilities of beta-lactamase inhibitors of the C-2- and C-3-substituted penicillin and cephalosporin sulfone families against OXA-1, extended-spectrum (OXA-10, OXA-14, and OXA-17), and carbapenemase-type (OXA-24/40) class D beta-lactamases. Three C-2-substituted penicillin sulfone compounds (JDB/LN-1-255, JDB/LN-III-26, and JDB/ASR-II-292) showed low K(i) values for the OXA-1 beta-lactamase (0.70 +/- 0.14 --> 1.60 +/- 0.30 microM) and demonstrated significant K(i) improvements compared to the C-3-substituted cephalosporin sulfone (JDB/DVR-II-214), tazobactam, and clavulanic acid. The C-2-substituted penicillin sulfones JDB/ASR-II-292 and JDB/LN-1-255 also demonstrated low K(i)s for the OXA-10, -14, -17, and -24/40 beta-lactamases (0.20 +/- 0.04 --> 17 +/- 4 microM). Furthermore, JDB/LN-1-255 displayed stoichiometric inactivation of OXA-1 (the turnover number, i.e., the partitioning of the initial enzyme inhibitor complex between hydrolysis and enzyme inactivation [t(n)] = 0) and t(n)s ranging from 5 to 8 for the other OXA enzymes. Using mass spectroscopy to study the intermediates in the inactivation pathway, we determined that JDB/LN-1-255 inhibited OXA beta-lactamases by forming covalent adducts that do not fragment. On the basis of the substrate and inhibitor kinetics of OXA-1, we constructed a model showing that the C-3 carboxylate of JDB/LN-1-255 interacts with Ser115 and Thr213, the R-2 group at C-2 fits between the space created by the long B9 and B10 beta strands, and stabilizing hydrophobic interactions are formed between the pyridyl ring of JDB/LN-1-255 and Val116 and Leu161. By exploiting conserved structural and mechanistic features, JDB/LN-1-255 is a promising lead compound in the quest for effective inhibitors of OXA-type beta-lactamases.
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Project description:Class D beta-lactamase-mediated resistance to beta-lactams has been increasingly reported during the last decade. Those enzymes also known as oxacillinases or OXAs are widely distributed among Gram negatives. Genes encoding class D beta-lactamases are known to be intrinsic in many Gram-negative rods, including Acinetobacter baumannii and Pseudomonas aeruginosa, but play a minor role in natural resistance phenotypes. The OXAs (ca. 150 variants reported so far) are characterized by an important genetic diversity and a great heterogeneity in terms of beta-lactam hydrolysis spectrum. The acquired OXAs possess either a narrow spectrum or an expanded spectrum of hydrolysis, including carbapenems in several instances. Acquired class D beta-lactamase genes are mostly associated to class 1 integron or to insertion sequences.
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