Crystal structures of the cadmium- and mercury-substituted metallo-beta-lactamase from Bacteroides fragilis.

The metallo-beta-lactamases require zinc or cadmium for hydrolyzing beta-lactam antibiotics and are inhibited by mercurial compounds. To data, there are no clinically useful inhibitors of this class of enzymes. The crystal structure of the Zn(2+)-bound enzyme from Bacteroides fragilis contains a binuclear zinc center in the active site. A hydroxide, coordinated to both zinc atoms, is proposed as the moiety that mounts the nucleophilic attack on the carbonyl carbon atom of the beta-lactam ring. To study the metal coordination further, the crystal structures of a Cd(2+)-bound enzyme and of an Hg(2+)-soaked zinc-containing enzyme have been determined at 2.1 A and 2.7 A, respectively. Given the diffraction resolution, the Cd(2+)-bound enzyme exhibits the same active-site architecture as that of the Zn(2+)-bound enzyme, consistent with the fact that both forms are enzymatically active. The 10-fold reduction in activity of the Cd(2+)-bound molecule compared with the Zn(2+)-bound enzyme is attributed to fine differences in the charge distribution due to the difference in the ionic radii of the two metals. In contrast, in the Hg(2+)-bound structure, one of the zinc ions, Zn2, was ejected, and the other zinc ion, Zn1, remained in the same site as in the 2-Zn(2+)-bound structure. Instead of the ejected zinc, a mercury ion binds between Cys 104 and Cys 181, 4.8 A away from Zn1 and 3.9 A away from the site where Zn2 is located in the 2-Zn(2+)-bound molecule. The perturbed binuclear metal cluster explains the inactivation of the enzyme by mercury compounds.     

Mutations of endo-beta-N-acetylglucosaminidase H active site residueAs sp130 anG glu132: activities and conformations

Endo-beta-N-acetylglucosaminidase H hydrolyzes the beta-(1-4)-glycosidic link of the N,N'-diacetylchitobiose core of high-mannose and hybrid asparagine-linked oligosaccharides. Seven mutants of the active site residues, Asp130 and Glu132, have been prepared, assayed, and crystallized. They include single site mutants of each residue to the corresponding amide, to Ala and to the alternate acidic residue, and to the double amide mutant. The mutants of Asp130 are more active than the corresponding Glu132 mutants, consistent with the assignment of the latter residue as the primary catalytic residue. The amide mutants are more active than the alternate acidic residue mutants, which in turn are more active than the Ala mutants. The structures of the Asn mutant of Asp130 and the double mutant are very similar to that of the wild-type enzyme. Several residues surrounding the mutated residues, including some that form part of the core of the beta-barrel and especially Tyr168 and Tyr244, adopt a very different conformation in the structures of the other two mutants of Asp130 and in the Asp mutant of Glu132. The results show that the residues in the upper layers of the beta-barrel can organize into two very distinct packing arrangements that depend on subtle electrostatic and steric differences and that greatly affect the geometry of the substrate-binding cleft. Consequently, the relative activities of several of the mutants are defined by structural changes, leading to impaired substrate binding, in addition to changes in functionality.     

In vivo folding of recombinant metallo-beta-lactamase L1 requires the presence of Zn(II)

Metallo-beta-lactamase L1, secreted by pathogenic Stenotrophomonas maltophilia, is a dinuclear Zn(II)-containing enzyme that hydrolyzes almost all known penicillins, cephalosporins, and carbapenems. The presence of Zn(II) ions in both metal binding sites is essential for full enzymatic activity; however, the mechanism of physiological metal incorporation is unknown. To probe metal incorporation, L1 was over-expressed in minimal media with (mmL1+Zn) and without (mmL1-Zn) Zn(II) added to the media, and the resulting proteins were purified and characterized. The mmL1+Zn sample was bound by a Q-Sepharose column, exhibited steady-state kinetic properties, bound Zn(II), existed as a tetramer, and yielded fluorescence emission and CD spectra similar to L1 overexpressed in rich media. On the other hand, the mmL1-Zn sample did not bind to a Q-Sepharose column, and gel filtration studies demonstrated that this protein was monomeric. The mmL1-Zn sample exhibited a lower kcat value, bound less Zn(II), and yielded fluorescence emission and CD spectra consistent with this enzyme being folded improperly. Taken together, these data demonstrate that the proper folding of L1 requires the presence of Zn(II) and suggest that in vitro, thermodynamic metal binding studies do not accurately reflect physiological metal incorporation into L1.     

Trapping the tetrahedral intermediate in the alkaline phosphatase reaction by substitution of the active site serine with threonine

We report here the construction of a mutant version of Escherichia coli alkaline phosphatase (AP) in which the active site Ser was replaced by Thr (S102T), in order to investigate whether the enzyme can utilize Thr as the nucleophile and whether the rates of the critical steps in the mechanism are altered by the substitution. The mutant AP with Thr at position 102 exhibited an approximately 4000-fold decrease in k(cat) along with a small decrease in Km. The decrease in catalytic efficiency of approximately 2000-fold was a much smaller drop than that observed when Ala or Gly were substituted at position 102. The mechanism by which Thr can substitute for Ser in AP was further investigated by determining the X-ray structure of the S102T enzyme in the presence of the Pi (S102T_Pi), and after soaking the crystals with substrate (S102T_sub). In the S102T_Pi structure, the Pi was coordinated differently with its position shifted by 1.3 A compared to the structure of the wild-type enzyme in the presence of Pi. In the S102T_sub structure, a covalent Thr-Pi intermediate was observed, instead of the expected bound substrate. The stereochemistry of the phosphorus in the S102T_sub structure was inverted compared to the stereochemistry in the wild-type structure, as would be expected after the first step of a double in-line displacement mechanism. We conclude that the S102T mutation resulted in a shift in the rate-determining step in the mechanism allowing us to trap the covalent intermediate of the reaction in the crystal.     

Grafting a new metal ligand in the cocatalytic site of B. cereus metallo-beta-lactamase: structural flexibility without loss of activity

Metallo-beta-lactamases are zinc enzymes able to hydrolyze the four-membered ring of beta-lactam antibiotics, representing one of the latest generations of beta-lactamases. These enzymes belong to the zinc metallo-hydrolase family of the beta-lactamase fold. Enzymes belonging to this family have a bimetallic active site whose structure varies among different members by point substitutions of the metal ligands. In this work, we have grafted new metal ligands into the metal binding site of BcII from Bacillus cereus that mimic the ligands present in other members of this superfamily. We have characterized spectroscopically and modeled the structure of the redesigned sites, which differ substantially from the wild-type enzyme. Despite the changes introduced in the active site, the mutant enzymes retain almost full activity. These results shed some light on the possible evolutionary origin of these metalloenzymes.     

Effect of the modification of the processing site of <Emphasis Type="Italic">Bacillus intermedius</Emphasis> glutamyl endopeptidase on the production of active enzyme by <Emphasis Type="Italic">Bacillus subtilis</Emphasis> cells

A site-directed modification at position P1 of the processing site of Bacillus intermedius glutamyl endopeptidase was carried out. Variants of the protease gene were obtained that correspond to the protein with an Ala, Asn, Ser, or Glu residue at this position substituted for the Lys residue. The residue in the P1 position of the processing site was shown to affect substantially the production of the active enzyme; however, none of the mutations leads to the complete termination of active protein production by cells.     

Structural characterization of the GSK-3beta active site using selective and non-selective ATP-mimetic inhibitors

GSK-3beta is a regulatory serine/threonine kinase with a plethora of cellular targets. Consequently, selective small molecule inhibitors of GSK-3beta may have a variety of therapeutic uses including the treatment of neurodegenerative diseases, type II diabetes and cancer. In order to characterize the active site of GSK-3beta, we determined crystal structures of unphosphorylated GSK-3beta in complex with selective and non-selective ATP-mimetic inhibitors. Analysis of the inhibitors' interactions with GSK-3beta in the structures reveals how the enzyme can accommodate a number of diverse molecular scaffolds. In addition, a conserved water molecule near Thr138 is identified that can serve a functional role in inhibitor binding. Finally, a comparison of the interactions made by selective and non-selective inhibitors highlights residues on the edge of the ATP binding-site that can be used to obtain inhibitor selectivity. Information gained from these structures provides a promising route for the design of second-generation GSK-3beta inhibitors.     

Modeling of the metallo-beta-lactamase from B. fragilis: structural and dynamic effects of inhibitor binding

The structure and dynamics of an inhibitor-bound complex of the metallo-beta-lactamase from Bacteroides fragilis are studied by using molecular dynamics. A search of the conformational space was performed to obtain three distinct models of the complex, which were then subjected to solvated molecular dynamics. A solvated molecular dynamics study of the apo protein was performed to serve as a baseline for comparison with the bound simulations. We find loop conformation changes due to binding as well as a decrease in flexibility of the protein as a whole and especially in the major loop of the beta-lactamase. We report the structural and dynamical features of the inhibitor-bound and apo models, as well as experimentally measurable quantities, which should be capable of distinguishing the two binding modes we have determined.     

Functional consequences of mutations at the allosteric interface in hetero- and homo-hemoglobin tetramers.

A seminal difference exists between the two types of chains that constitute the tetrameric hemoglobin in vertebrates. While alpha chains associate weakly into dimers, beta chains self-associate into tightly assembled tetramers. While heterotetramers bind ligands cooperatively with moderate affinity, homotetramers bind ligands with high affinity and without cooperativity. These characteristics lead to the conclusion that the beta 4 tetramer is frozen in a quaternary R-state resembling that of liganded HbA. X-ray diffraction studies of the liganded beta 4 tetramers and molecular modeling calculations revealed several differences relative to the native heterotetramer at the "allosteric" interface (alpha 1 beta 2 in HbA) and possibly at the origin of a large instability of the hypothetical deoxy T-state of the beta 4 tetramer. We have studied natural and artificial Hb mutants at different sites in the beta chains responsible for the T-state conformation in deoxy HbA with the view of restoring a low ligand affinity with heme-heme interaction in homotetramers. Functional studies have been performed for oxygen equilibrium binding and kinetics after flash photolysis of CO for both hetero- and homotetramers. Our conclusion is that the "allosteric" interface is so precisely tailored for maintaining the assembly between alpha beta dimers that any change in the side chains of beta 40 (C6), beta 99 (G1), and beta 101 (G3) involved in the interface results in increased R-state behavior. In the homotetramer, the mutations at these sites lead to the destabilization of the beta 4 hemoglobin and the formation of lower affinity noncooperative monomers.     

A disulfide bridge near the active site of carbapenem-hydrolyzing class A β-lactamases might explain their unusual substrate profile

Bacterial resistance to β-lactam antibiotics, a clinically worrying and recurrent problem, is often due to the production of β-lactamases, enzymes that efficiently hydrolyze the amide bond of the β-lactam nucleus. Imipenem and other carbapenems escape the activity of most active site serine β-lactamases and have therefore become very popular drugs for antibacterial chemotherapy in the hospital environment. Their usefulness is, however, threatened by the appearance of new β-lactamases that efficiently hydrolyze them. This study is focused on the structure and properties of two recently described class A carbapenemases, produced by Serratia marcescens and Enterobacter cloacae strains and leads to a better understanding of the specificity of β-lactamases. In turn, this will contribute to the design of better antibacterial drugs. Three-dimensional models of the two class A carbapenemases were constructed by homology modeling. They suggested the presence, near the active site of the enzymes, of a disulfide bridge (C69-C238) whose existence was experimentally confirmed. Kinetic parameters were measured with the purified Sme-1 carbapenemase, and an attempt was made to explain its specific substrate profile by analyzing the structures of minimized Henri-Michaelis complexes and comparing them to those obtained for the “classical” TEM-1 β-lactamase. The peculiar substrate profile of the carbapenemases appears to be strongly correlated with the presence of the disulfide bridge between C69 and C238. Proteins 27:47–58 © 1997 Wiley-Liss, Inc.     

Nucleotide Sequence of the Gene Encoding β-Isopropylmalate Dehydrogenase (leuB) from Bacteroides fragilis

The complete nucleotide sequence of the gene (leuB) coding for β-isopropylmaiate dehydrogenase of Bacteroides fragilis was determined. An open reading frame of 1,061 nucleotides was detected that could encode a polypeptide of 353 amino acid residues with a calculated molecular mass of 39,179 Da. The deduced amino acid sequence of the β-isopropylmalate dehydrogenase from B. fragilis showed substantial sequence similarity with the β-isopropylmalate dehydrogenases from other bacteria.     

The co-crystal structure of unliganded bovine α-thrombin and prethrombin-2: Movement of the Tyr-Pro-Pro-Trp segment and active site residues upon ligand binding

Unliganded bovine α-thrombin and prethrombin-2 have been co-crystallized, in space group P2₁2₁2, using either ammonium sulfate or polyethylene glycol 2000 (PEG2K), and their structures determined at 2.2 Å and 2.3 Å, respectively. Initial phases were determined by molecular replacement and refined using XPLOR to final R factors of 0.187 (R_free = 0.255) and 0.190 (R_free = 0.282) for the salt and PEG2K models, respectively. The apo-enzyme form of bovine α-thrombin shows dramatic shifts in placement for the Tyr-Pro-Pro-Trp segment, for Glu-192, and for the catalytic residues His-57 and Ser-195, when compared to 4 thrombin complexes representing different states of catalysis, namely (1) the Michaelis complex (residues 7-19 of fibrinogen Aa with a non-cleavable scissile bond), (2) enzyme-inhibitor complex (D-Phe-Pro-Arg chloromethylketone), (3) enzyme product complex (residues 7-16 of fibrinopeptide A), and (4) the exosite complex (residues 53-64 of hirudin). The structures of bovine and human prethrombin-2 are generally similar to one another (RMS deviation of 0.68 8,) but differ significantly in the Arg-15/Ile-16 cleavage region and in the three activation domains, which are disordered in bovine prethrombin-2, analogous to that seen for trypsinogen.     

Structural analysis of the zinc hydroxide–Thr-199–Glu-106 hydrogen-bond network in human carbonic anhydrase II

The singnificance of the zinc hydroxide–Thr-199–Glu-106 hydrogen-bond network in the active site of human carbonic anhydrase II has been examined by X-ray crystallographic analyses of site-specific mutants. Mutants with Ala-199 and Ala-106 or Gln-106 have low catalytic activities, while a mutant with Asp-106 has almost full CO₂ hydration activity. The structures of these four mutants, as well as that of the bicarbonate complex of the mutant with Ala-199, have been determined at 1.7 to 2.2 Å resolution. Removal of the γ atoms of residue 199 leads to distorted tetrahedral geometry at the zine ion, and a catalytically important zinc-bound water molecule has moved towards Glu-106. In the bicarbonate complex of the mutant with Ala-199 one oxygen atom from bicarbonate binds to zinc without displacing this water molecule. Tetrahedral coordination geometries are retained in the mutants at position 106. The mutants with Ala-106 and Gln-106 have a zinc-bound sulfate ion, whereas this sulfate site is only partially occupied in the mutant with Asp-106. The hydrogen-bond network seems to be “reversed” in the mutants with Ala-106 and Gln-106. The network is preserved as in native enzyme in the mutant with Asp-106 but the side chain of Asp-106 is more extended than that of Glu-106 in the native enzyme. These results illustrate the importance of Glu-106 and Thr-199 for controlling the precise coordination geometry of the zinc ion and its ligand preferences with results in an optimal orientation of a zine-bound hydroxide ion for an attack on the CO₂ substrate. © 1993 Wiley-Liss, Inc.     

Structural basis of increased resistance to thermal denaturation induced by single amino acid substitution in the sequence of β-glucosidase A from Bacillus polymyxa

The increasing development of the biotechnology industry demands the design of enzymes suitable to be used in conditions that often require broad resistance against adverse conditions. β-glucosidase A from Bacillus polymyxa is an interesting model for studies of protein engineering. This is a well-characterized enzyme, belonging to glycosyl hydrolase family 1. Its natural substrate is cellobiose, but is also active against various artificial substrates. In its native state has an octameric structure. Its subunit conserves the general (α/β)₈ barrel topology of its family, with the active site being in a cavity defined along the axis of the barrel. Using random-mutagenesis, we have identified several mutations enhancing its stability and it was found that one them, the E96K substitution, involved structural changes. The crystal structure of this mutant has been determined by X-ray diffraction and compared with the native structure. The only difference founded between both structures is a new ion pair linking Lys96 introduced at the N-terminus of helix α2, to Asp28, located in one of the loops surrounding the active-site cavity. The new ion pair binds two segments of the chain that are distant in sequence and, therefore, this favorable interaction must exert a determinant influence in stabilizing the tertiary structure. Furthermore, analysis of the crystallographic isotropic temperature factors reveals that, as a direct consequence of the introduced ion pair, an unexpected decreased mobility of secondary structure units of the barrel which are proximal to the site of mutation is observed. However, this effect is observed only in the surrounding of one of the partners forming the salt bridge and not around the other. These results show that far-reaching effects can be achieved by a single amino acid replacement within the protein structure. Consequently, the identification and combination of a few single substitutions affecting stability may be sufficient to obtain a highly resistant enzyme, suitable to be used under extreme conditions. Proteins 33:567–576, 1998. © 1998 Wiley-Liss, Inc.     

Structural effects of the active site mutation cysteine to serine in Bacillus cereus zinc-beta-lactamase

Beta-lactamases are involved in bacterial resistance. Members of the metallo-enzyme class are now found in many pathogenic bacteria and are becoming thus of major clinical importance. Despite the availability of Zn-beta-lactamase X-ray structures their mechanism of action is still unclear. One puzzling observation is the presence of one or two zincs in the active site. To aid in assessing the role of zinc content in beta-lactam hydrolysis, the replacement by Ser of the zinc-liganding residue Cys168 in the Zn-beta-lactamase from Bacillus cereus strain 569/H/9 was carried out: the mutant enzyme (C168S) is inactive in the mono-Zn form, but active in the di-Zn form. The structure of the mono-Zn form of the C168S mutant has been determined at 1.85 A resolution. Ser168 occupies the same position as Cys168 in the wild-type enzyme. The protein residues mostly affected by the mutation are Asp90-Arg91 and His210. A critical factor for the activity of the mono-Zn species is the distance between Asp90 and the Zn ion, which is controlled by Arg91: a slight movement of Asp90 impairs catalysis. The evolution of a large superfamily including Zn-beta-lactamases suggests that they may not all share the same mechanism.     

Structural insights into the design of inhibitors for the L1 metallo-beta-lactamase from Stenotrophomonas maltophilia

One mechanism by which bacteria can escape the action of β-lactam antibiotics is the production of metallo-β-lactamases. Inhibition of these enzymes should restore the action of these widely used antibiotics. The tetrameric enzyme L1 from Stenotrophomonas maltophilia was used as a model system to determine a series of high-resolution crystal structures of apo, mono and bi-metal substituted proteins as well as protein-inhibitor complexes. Unexpectedly, although the apo structure revealed only few significant structural differences from the holo structure, some inhibitors were shown to induce amino acid side-chain rotations in the tightly packed active site. Moreover, one inhibitor employs a new binding mode in order to interact with the di-zinc center. This structural information could prove essential in the process of elucidation of the mode of interaction between a putative lead compound and metallo-β-lactamases, one of the main steps in structure-based drug design.     

Chemical modification of the RTEM-1 thiol beta-lactamase by thiol-selective reagents: evidence for activation of the primary nucleophile of the beta-lactamase active site by adjacent functional groups

The RTEM-1 thiol beta-lactamase (Sigal, I.S., Harwood, B.G., Arentzen, R., Proc. Natl. Acad. Sci. U.S.A. 79:7157-7160, 1982) is inactivated by thiol-selective reagents such as iodoacetamide, methyl methanethiosulfonate, and 4,4'-dipyridyldisulfide, which modify the active site thiol group. The pH-rate profiles of these inactivation reactions show that there are two nucleophilic forms of the enzyme, EH2 and EH, both of which, by analogy with the situation with cysteine proteinases, probably contain the active site nucleophile in the thiolate form. The pKa of the active site thiol is therefore shown by the data to be below 4.0. This low pKa is thought to reflect the presence of adjacent functionality which stabilizes the thiolate anion. The low nucleophilicity of the thiolate in both EH2 and EH, with respect to that of cysteine proteinases and model compounds, suggests that the thiolate of the thiol beta-lactamase is stabilized by two hydrogen-bond donors. One of these, of pKa greater than 9.0, is suggested to be the conserved and essential Lys-73 ammonium group, while the identity of the other group, of pKa around 6.7, is less clear, but may be the conserved Glu-166 carboxylic acid. beta-Lactamase activity is associated with the EH2 form, and thus the beta-lactamase active site is proposed to contain one basic or nucleophilic group (the thiolate in the thiol beta-lactamase) and two acidic (hydrogen-bond donor) groups (one of which is likely to be the above-mentioned lysine ammonium group).     

Mutational analysis of the active site of indoleglycerol phosphate synthase from Escherichia coli.

Indoleglycerol phosphate synthase catalyzes the ring closure of 1-(2-carboxyphenylamino)-1-deoxyribulose 5''-phosphate to indoleglycerol phosphate, the fifth step in the pathway of tryptophan biosynthesis from chorismate. Because chemical synthesis of indole derivatives from arylamino ketones requires drastic solvent conditions, it is interesting by what mechanism the enzyme catalyzes the same condensation reaction. Seven invariant polar residues in the active site of the enzyme from Escherichia coli have been mutated directly or randomly, to identify the catalytically essential ones. A strain of E. coli suitable for selecting and classifying active mutants by functional complementation was constructed by precise deletion of the trpC gene from the genome. Judged by growth rates of transformants on selective media, mutants with either S58 or S60 replaced by alanine were indistinguishable from the wild-type, but R186 replaced by alanine was still partially active. Saturation random mutagenesis of individual codons showed that E53 was partially replaceable by aspartate and cysteine, whereas K114, E163, and N184 could not be replaced by any other residue. Partially active mutant proteins were purified and their steady-state kinetic and inhibitor binding constants determined. Their relative catalytic efficiencies paralleled their relative complementation efficiencies. These results are compatible with the location of the essential residues in the active site of the enzyme and support a chemically plausible catalytic mechanism. It involves two enzyme-bound intermediates and general acid-base catalysis by K114 and E163 with the support of E53 and N184.