1.85 Å Resolution Structure of the ZincII β-Lactamase from Bacillus cereus

1998 ◽  
Vol 54 (3) ◽  
pp. 313-323 ◽  
Author(s):  
Andrea Carfi ◽  
Emile Duée ◽  
Moreno Galleni ◽  
Jean-Marie Frère ◽  
Otto Dideberg
Keyword(s):  
Bacillus Cereus ◽  
Active Site ◽  
Metal Ion ◽  
Wide Spectrum ◽  
Zinc Ion ◽  
Beta Lactamases ◽  
Carbonate Ion ◽  
Class B ◽  
Bivalent Metal Ions ◽  
Using Data

Class B \beta-lactamases are wide spectrum enzymes which require bivalent metal ions for activity. The structure of the class B zinc-ion-dependent β-lactamase from Bacillus cereus (BCII) has been refined at 1.85 Å resolution using data collected on cryocooled crystals (100 K). The enzyme from B. cereus has a molecular mass of 24 946 Da and is folded into a \beta-sandwich structure with helices on the external faces. The active site is located in a groove running between the two \beta-sheets [Carfi et al. (1995). EMBO J. 14, 4914–4921]. The 100 K high-resolution BCII structure shows one fully and one partially occupied zinc site. The zinc ion in the fully occupied site (the catalytic zinc) is coordinated by three histidines and one water molecule. The second zinc ion is at 3.7 Å from the first one and is coordinated by one histidine, one cysteine, one aspartate and one unknown molecule (which is most likely to be a carbonate ion). In the B. cereus zinc \beta-lactamase the affinity for the second metal ion is low at the pH of crystallization (Kd = 25 mM, 293 K; [Baldwin et al. (1978). Biochem. J. 175, 441–447] and the dissociation constant of the second zinc ion thus apparently decreased at the cryogenic temperature. In addition, the structure of the apo enzyme was determined at 2.5 Å resolution. The removal of the zinc ion by chelating agents causes small changes in the active-site environment.

scholarly journals The Structure of the Dizinc Subclass B2 Metallo-β-Lactamase CphA Reveals that the Second Inhibitory Zinc Ion Binds in the Histidine Site

2009 ◽  
Vol 53 (10) ◽  
pp. 4464-4471 ◽  
Author(s):  
Carine Bebrone ◽  
Heinrich Delbrück ◽  
Michaël B. Kupper ◽  
Philipp Schlömer ◽  
Charlotte Willmann ◽  
...  
Keyword(s):  
Active Site ◽  
Aeromonas Hydrophila ◽  
Zinc Ion ◽  
Amide Bond ◽  
Zinc Ions ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
X Ray ◽  
Metal Ligands ◽  
Class B

ABSTRACT Bacteria can defend themselves against β-lactam antibiotics through the expression of class B β-lactamases, which cleave the β-lactam amide bond and render the molecule harmless. There are three subclasses of class B β-lactamases (B1, B2, and B3), all of which require Zn2+ for activity and can bind either one or two zinc ions. Whereas the B1 and B3 metallo-β-lactamases are most active as dizinc enzymes, subclass B2 enzymes, such as Aeromonas hydrophila CphA, are inhibited by the binding of a second zinc ion. We crystallized A. hydrophila CphA in order to determine the binding site of the inhibitory zinc ion. X-ray data from zinc-saturated crystals allowed us to solve the crystal structures of the dizinc forms of the wild-type enzyme and N220G mutant. The first zinc ion binds in the cysteine site, as previously determined for the monozinc form of the enzyme. The second zinc ion occupies a slightly modified histidine site, where the conserved His118 and His196 residues act as metal ligands. This atypical coordination sphere probably explains the rather high dissociation constant for the second zinc ion compared to those observed with enzymes of subclasses B1 and B3. Inhibition by the second zinc ion results from immobilization of the catalytically important His118 and His196 residues, as well as the folding of the Gly232-Asn233 loop into a position that covers the active site.

scholarly journals A thiono-β-lactam substrate for the β-lactamase II of Bacillus cereus. Evidence for direct interaction between the essential metal ion and substrate

1989 ◽  
Vol 258 (3) ◽  
pp. 765-768 ◽  
Author(s):  
B P Murphy ◽  
R F Pratt
Keyword(s):  
Bacillus Cereus ◽  
Active Site ◽  
Metal Ion ◽  
Direct Interaction ◽  
Beta Lactamase ◽  
Beta Lactam ◽  
Enzyme Active Site ◽  
Lactam Carbonyl ◽  
Beta Lactamase Ii ◽  
Hydrolysis Of

An 8-thionocephalosporin was shown to be a substrate of the beta-lactamase II of Bacillus cereus, a zinc metalloenzyme. Although it is a poorer substrate, as judged by the Kcat./Km parameter, than the corresponding 8-oxocephalosporin, the discrimination against sulphur decreased when the bivalent metal ion in the enzyme active site was varied in the order Mn2+ (the manganese enzyme catalysed the hydrolysis of the oxo compound but not that of the thiono compound), Zn2+, Co2+ and Cd2+. This result is taken as evidence for kinetically significant direct contact between the active-site metal ion of beta-lactamase II and the beta-lactam carbonyl heteroatom. No evidence was obtained, however, for accumulation of an intermediate with such co-ordination present.

scholarly journals Learning from oligosaccharide soaks of crystals of an AA13 lytic polysaccharide monooxygenase: crystal packing, ligand binding and active-site disorder

2017 ◽  
Vol 73 (1) ◽  
pp. 64-76 ◽  
Author(s):  
Kristian E. H. Frandsen ◽  
Jens-Christian Navarro Poulsen ◽  
Morten Tovborg ◽  
Katja S. Johansen ◽  
Leila Lo Leggio
Keyword(s):  
Active Site ◽  
Metal Ion ◽  
Crystal Packing ◽  
Zinc Ion ◽  
Glycoside Hydrolases ◽  
Zinc Ions ◽  
Lytic Polysaccharide Monooxygenase ◽  
Cell Parameters ◽  
Glycosidic Bonds ◽  
High Zinc

Lytic polysaccharide monooxygenases (LPMOs) are a class of copper-dependent enzymes discovered within the last ten years. They oxidatively cleave polysaccharides (chitin, lignocellulose, hemicellulose and starch-derived), presumably making recalcitrant substrates accessible to glycoside hydrolases. Recently, the first crystal structure of an LPMO–substrate complex was reported, giving insights into the interaction of LPMOs with β-linked substrates (Frandsenet al., 2016). The LPMOs acting on α-linked glycosidic bonds (family AA13) display binding surfaces that are quite different from those of LPMOs that act on β-linked glycosidic bonds (families AA9–AA11), as revealed from the first determined structure (Lo Leggioet al., 2015), and thus presumably the AA13s interact with their substrate in a distinct fashion. Here, several new structures of the same AA13 enzyme,Aspergillus oryzaeAA13, are presented. Crystals obtained in the presence of high zinc-ion concentrations were used, as they can be obtained more reproducibly than those used to refine the deposited copper-containing structure. One structure with an ordered zinc-bound active site was solved at 1.65 Å resolution, and three structures from crystals soaked with maltooligosaccharides in solutions devoid of zinc ions were solved at resolutions of up to 1.10 Å. Despite similar unit-cell parameters, small rearrangements in the crystal packing occur when the crystals are depleted of zinc ions, resulting in a more occluded substrate-binding surface. In two of the three structures maltooligosaccharide ligands are bound, but not at the active site. Two of the structures presented show a His-ligand conformation that is incompatible with metal-ion binding. In one of these structures this conformation is the principal one (80% occupancy), giving a rare atomic resolution view of a substantially misfolded enzyme that is presumably rendered inactive.

Interaction of β-lactamases I and II from Bacillus cereus with semisynthetic cephamycins. Kinetic studies

1991 ◽  
Vol 279 (1) ◽  
pp. 111-114 ◽  
Author(s):  
J Martin Villacorta ◽  
P Arriaga ◽  
J Laynez ◽  
M Menendez
Keyword(s):  
Bacillus Cereus ◽  
Kinetic Studies ◽  
Beta Lactamase ◽  
Beta Lactam ◽  
Beta Lactamases ◽  
Class A ◽  
Rate Determining Step ◽  
Class B ◽  
Lactam Ring ◽  
Beta Lactamase Ii

The influence of C-6 alpha- or C-7 alpha-methoxylation of the beta-lactam ring in the catalytic action of class A and B beta-lactamases has been investigated. For this purpose the kinetic behaviour of beta-lactamases I (class A) and II (class B) from Bacillus cereus was analysed by using several cephamycins, moxalactam, temocillin and related antibiotics. These compounds behaved as poor substrates for beta-lactamase II, with high Km values and very low catalytic efficiencies. In the case of beta-lactamase I, the substitution of a methoxy group for a H atom at C-7 alpha or C-6 alpha decreased the affinity of the substrates for the enzyme. Furthermore, the acylation of cephamycins was completely blocked, whereas that of penicillins was slowed down by a factor of 10(4)-10(5), acylation being the rate-determining step of the process.

DIFFERENT PROTONATION STATES OF THE BACILLUS CEREUS BINUCLEAR ZINC METALLO-β-LACTAMASE ACTIVE SITE STUDIED BY COMBINED QUANTUM MECHANICAL AND MOLECULAR MECHANICAL SIMULATIONS

2002 ◽  
Vol 01 (01) ◽  
pp. 69-80 ◽  
Author(s):  
WEI GU ◽  
JIANG ZHU ◽  
HAIYAN LIU
Keyword(s):  
Water Molecule ◽  
Bacillus Cereus ◽  
Ab Initio ◽  
Active Site ◽  
Density Functional ◽  
Tight Binding ◽  
Zinc Ion ◽  
Empirical Method ◽  
Quantum Mechanical ◽  
Stable Arrangement

Three different protonation states of the active site of the Bacillus cereus zinc-β-lactamase in its binuclear form are studied using combined quantum mechanics/molecular mechanics molecular dynamics simulations. The reliability of the quantum mechanical model, the self-consistent-charge density-functional-based tight binding method, in describing the zinc centers are tested through comparisons with ab initio quantum mechanical results. We found that this model gave relatively accurate results for structures and performed much better than the MNDO type semi-empirical method for the particular systems. The enzyme simulations suggested that when the overall charge of the active site is +1, i.e., both Asp90 and Wat1 (a water molecule coordinated with the first zinc ion) deprotonated, the second zinc ion is coordinated with Asp90 and Wat1, and a second water molecule cannot coordinate with the second zinc ion. When the overall charge is +2, i.e., either Asp90 or Wat1 protonated, Asp90 and Wat1 form a stable hydrogen bond. Depending on the proton being on Asp90 or on Wat1, the active site structure produced by the simulations is either similar to molecule A or to molecule B, both contained in the same crystal structure that has two enzyme molecules in a single asymmetric unit. The simulations of the +2 charge states also reproduced the experimentally observed "loose" coordination around the second zinc for the Bacillus Cereus enzyme. Based on the simulations and a gas phase potential energy surface scanning using ab initio model, we argue that the penta-coordination around the second zinc ion is not a stable arrangement. Mechanistic implications of these results are discussed.

Active sites of β-lactamases from Bacillus cereus

1980 ◽  
Vol 289 (1036) ◽  
pp. 333-344 ◽  
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Amino Acid ◽  
Bacillus Cereus ◽  
Active Site ◽  
Metal Ion ◽  
Active Sites ◽  
Extracellular Enzyme ◽  
Thiol Group ◽  
The Other ◽  
Site Group

There are two extracellular β-lactamases produced by Bacillus cereus 569. One of these enzymes, β-lactamase I, is inactivated by 6-β-bromopenicillanic acid: the site of reaction is serine-44. This is a conserved amino acid residue in the other β-lactamases whose structures have been determined, and it becomes a good candidate for an active-site group in these enzymes. The inactivation may involve a rearrangement leading to a dihydrothiazine. The other extracellular enzyme produced by B. cereus , β- lactamase II, is exceptional in requiring metal ions for activity. The Zn II and Co II enzymes (the former is more active) have been studied by nuclear magnetic resonance, and by absorption spectroscopy. The groups that bind the metal ion required for activity are three histidine residues and the enzyme’s sole thiol group.

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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