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In this article we analyze the reasons for catalytic promiscuity of a type VIII esterase with β-lactamase fold and the ability to cleave β-lactams. We compared the structure of this enzyme to those of an esterase of the same type without any lactamase ability, an esterase with moderate lactamase ability, and a class C β-lactamase with similar fold. Our results show that for these enzymes, the difference in the substrate specificity is sterically driven., (Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

One often-cited weakness of biocatalysis is the lack of mirror-image enzymes for the formation of either enantiomer of a product in asymmetric synthesis. Enantiocomplementary enzymes exist as the solution to this problem in nature. These enzyme pairs, which catalyze the same reaction but favor opposite enantiomers, are not mirror-image molecules; however, they contain active sites that are functionally mirror images of one another. To create mirror-image active sites, nature can change the location of the binding site and/or the location of key catalytic groups. In this Minireview, X-ray crystal structures of enantiocomplementary enzymes are surveyed and classified into four groups according to how the mirror-image active sites are formed.

Esterase EstB from Burkholderia gladioli, which belongs to a family of esterases related to beta-lactamases and DD-peptidases was evolved for increased stability and simultaneously maintaining high cephalosporin C deacetylation activity. Random mutagenesis PCR was used to generate up to 5 aa substitutions per gene. A newly designed colony filter-screening assay, which was based on pH change after deacetylation of cephalosporin C in presence of DMF was established. In a first evolution round employing random mutagenesis, which included about 10(6) mutants, a set of interesting mutants was isolated. Distinct mutations identified as significant for stability were combined by a rational recombination step and the resulting recombinant was further evolved by an additional random mutagenesis round. After screening an additional 10(5) clones, it was possible to isolate a variant of EstB having more than 100-fold better activity in reactions containing 35% DMF. This mutant also showed a high increase in temperature stability (T(m) was raised by 13 degrees C) and retained high activity towards cephalosporin C under standard assay conditions. The molecular effects of mutations found in random mutants are discussed in view of the three-dimensional structure of wild-type EstB.

The assembly of inflammatory lesions in rheumatoid arthritis is highly regulated and typically leads to the formation of lymphoid follicles with germinal center (GC) reactions. We used microdissection of such extranodal follicles to analyze the colonizing T cells. Although the repertoire of follicular T cells was diverse, a subset of T cell receptor (TCR) sequences was detected in multiple independent follicles and not in interfollicular zones, suggesting recognition of a common antigen. Unexpectedly, the majority of shared TCR sequences were from CD8 T cells that were highly enriched in the synovium and present in low numbers in the periphery. To examine their role in extranodal GC reactions, CD8 T cells were depleted in human synovium-SCID mouse chimeras. Depletion of synovial CD8 T cells caused disintegration of the GC-containing follicles. In the absence of CD8 T cells, follicular dendritic cells disappeared, production of lymphotoxin-alpha1beta2 markedly decreased, and immunoglobulin (Ig) secretion ceased. Immunohistochemical studies demonstrated that these CD8 T cells accumulated at the edge of the mantle zone. Besides their unique localization, they were characterized by the production of interferon (IFN)-gamma, lack of the pore-forming enzyme perforin, and expression of CD40 ligand. Perifollicular IFN-gamma+ CD8 T cells were rare in secondary lymphoid tissues but accounted for the majority of IFN-gamma+ cells in synovial infiltrates. We propose that CD8+ T cells regulate the structural integrity and functional activity of GCs in ectopic lymphoid follicles.

Esterases form a diverse class of enzymes of largely unknown physiological role. Because many drugs and pesticides carry ester functions, the hydrolysis of such compounds forms at least one potential biological function. Carboxylesterases catalyze the hydrolysis of short chain aliphatic and aromatic carboxylic ester compounds. Esterases, D-alanyl-D-alanine-peptidases (DD-peptidases) and beta-lactamases can be grouped into two distinct classes of hydrolases with different folds and topologically unrelated catalytic residues, the one class comprising of esterases, the other one of beta-lactamases and DD-peptidases. The chemical reactivities of esters and beta-lactams towards hydrolysis are quite similar, which raises the question of which factors prevent esterases from displaying beta-lactamase activity and vice versa. Here we describe the crystal structure of EstB, an esterase isolated from Burkholderia gladioli. It shows the protein to belong to a novel class of esterases with homology to Penicillin binding proteins, notably DD-peptidase and class C beta-lactamases. Site-directed mutagenesis and the crystal structure of the complex with diisopropyl-fluorophosphate suggest Ser75 within the "beta-lactamase" Ser-x-x-Lys motif to act as catalytic nucleophile. Despite its structural homology to beta-lactamases, EstB shows no beta-lactamase activity. Although the nature and arrangement of active-site residues is very similar between EstB and homologous beta-lactamases, there are considerable differences in the shape of the active site tunnel. Modeling studies suggest steric factors to account for the enzyme's selectivity for ester hydrolysis versus beta-lactam cleavage.

Background: Transport of molybdenum into bacteria involves a high-affinity ABC transporter system whose expression is controlled by a repressor protein called ModE. While molybdate transport is tightly coupled to utilization in some bacteria, other organisms have molybdenum storage proteins. One class of putative molybdate storage proteins is characterized by a sequence consisting of about 70 amino acids (Mop). A tandem repeat of Mop sequences also constitutes the molybdate binding domain of ModE., Results: We have determined the crystal structure of the 7 kDa Mop protein from the methanol-utilizing anaerobic eubacterium Sporomusa ovata grown in the presence of molybdate and tungstate. The protein occurs as highly symmetric hexamers binding eight oxyanions. Each peptide assumes a so-called OB fold, which has previously also been observed in ModE. There are two types of oxyanion binding sites in Mo at the interface between two or three peptides. All oxyanion binding sites were found to be occupied by WO(4) rather than MoO(4)., Conclusions: The biological function of proteins containing only Mop sequences is unknown, but they have been implicated in molybdate homeostasis and molybdopterin cofactor biosynthesis. While there are few indications that the S. ovata Mop binds pterin, the structure suggests that only the type-1 oxyanion binding sites would be sufficiently accessible to bind a cofactor. The observed occupation of the oxyanion binding sites by WO(4) indicates that Mop might also be involved in controlling intracellular tungstate levels.

The 3D structures of complexes between the hydroxynitrile lyase from Hevea brasiliensis (Hb-HNL) and several substrate and/or inhibitor molecules, including trichloracetaldehyde, hexafluoracetone, acetone, and rhodanide, were determined by X-ray crystallography. The complex with trichloracetaldehyde showed a covalent linkage between the protein and the inhibitor, which had apparently resulted from nucleophilic attack of the catalytic Ser80-Ogamma. All other complexes showed the substrate or inhibitor molecule merely hydrogen bonded to the protein. In addition, the native crystal structure of Hb-HNL was redetermined at cryo-temperature and at room temperature, eliminating previous uncertainties concerning residual electron density within the active site, and leading to the observation of two conserved water molecules. One of them was found to be conserved in all complex structures and appears to have mainly structural significance. The other water molecule is conserved in all structures except for the complex with rhodanide; it is hydrogen bonded to the imidazole of the catalytic His235 and appears to affect the Hb-HNL catalyzed reaction. The observed 3D structural data suggest implications for the enzyme mechanism. It appears that the enzyme-catalyzed cyanohydrin formation is unlikely to proceed via a hemiacetal or hemiketal intermediate covalently attached to the enzyme, despite the observation of such an intermediate for the complex with trichloracetaldehyde. Instead, the data are consistent with a mechanism where the incoming substrate is activated by hydrogen bonding with its carbonyl oxygen to the Ser80 and Thr11 hydroxy groups. A hydrogen cyanide molecule subsequently replaces a water molecule and is deprotonated presumably by the His235 base. Deprotonation is facilitated by the proximity of the positive charge of the Lys236 side chain.

The X-ray crystal structure of native hydroxynitrile lyase from Hevea brasiliensis (Hb-HNL) has been determined at 1.1 A resolution. It refined to a final R of 11.5% for all data and an Rfree of 14.4%. The favorable data-to-parameter ratio at atomic resolution made the refinement of individual anisotropic displacement parameters possible. The data also allowed a clear distinction of the alternate orientations of all histidine and the majority of asparagine and glutamine side chains. A number of hydrogen atoms, including one on the imidazole of the mechanistically important His-235, became visible as peaks in a difference electron density map. The structure revealed a discretely disordered sidechain of Ser-80, which is part of the putative catalytic triad. Analysis of the anisotropy indicated an increased mobility of residues near the entrance to the active site and within the active site.

In rheumatoid synovitis, lymphocytes can be arranged in follicular structures resembling secondary lymphoid follicles. To understand the organizing principles of this ectopic lymphoid tissue, the cellular components contributing to synovial follicles were examined. In 9 of 24 synovial tissue biopsies, lymphoid aggregates were found consisting of CD4+ T cells and CD20+ B cells. In four of the nine patients, the follicular centers were occupied by CD23+ CD21+ cellular networks representing follicular dendritic cells involved in germinal center reactions. In five patients, CD23+ cells were absent from the centers of the aggregates, suggesting that fully developed germinal centers are generated in only a subset of patients. To identify factors involved in the regulation of the synovial microarchitecture, cell populations contributing to the follicles were quantified by digital image analysis of immunostained tissue and by flow cytometry of tissue-derived lymphocytes. Proportions of CD4+, CD20+, and CD68+ cell subsets were surprisingly invariant, irrespective of the presence or absence of CD23+ follicular dendritic cells. Instead, tissue biopsies with CD23+ germinal center-like regions could be distinguished from those with CD23- T cell-B cell aggregates by a fourfold increase in the frequency of tissue-infiltrating CD8+ T cells, a fraction of which expressed CD40 ligand (CD40L). The data suggest a previously unsuspected role of CD8+ lymphocytes in modulating germinal center formation and raise the possibility that CD8+ CD40L+ T cells are involved in aggravating pathologic immune responses in rheumatoid synovitis.

Objective: To identify the functional properties of CD4+ CD28- T cells, which accumulate and clonally expand in patients with rheumatoid arthritis (RA)., Methods: The gene expression of molecules involved in T cell effector functions was compared in CD4+ CD28- and CD4+ CD28+ T cell clones. The expression of differentially up-regulated genes was confirmed by flow cytometry of T cells and by 2-color immunohistochemistry of rheumatoid synovial tissue. Cytotoxicity of CD4+ CD28- T cells was tested by anti-CD3 redirected lysis of Fc receptor-positive target cells., Results: CD4+ CD28- T cell clones lacked messenger RNA for the CD40 ligand (CD40L) but transcribed the perforin gene. Perforin was also found in freshly isolated CD4+ CD28- peripheral blood lymphocytes from RA patients. CD4+ CD28-, but not CD4+ CD28+, T cell clones lysed Fc receptor-bearing target cells. CD4+ perforin-positive T cells were present in the synovial tissue, where their frequency correlated with the expansion of the CD4+ CD28- compartment in the periphery. Among tissue-infiltrating CD4+ T cells, only the CD40L-negative subset expressed perforin transcripts., Conclusion: Clonally expanded CD4+ CD28- T cells are functionally specialized for killing, while they lack the ability to provide B cell help. Tissue-infiltrating CD4+ T cells can be subdivided phenotypically and functionally into at least 2 distinct subsets based on their expression of perforin and CD40L. Because the expansion of CD4+ CD28- T cells is associated with extraarticular RA, T cell-mediated cytotoxicity may be particularly important in these most severe complications of RA.

Aberrations in the T cell repertoire with the emergence of oligoclonal populations have been described in patients with rheumatoid arthritis (RA). However, the extent of the repertoire perturbations as well as the underlying mechanisms are not known. We now have examined the diversity of the peripheral CD4 T cell repertoire by determining the frequencies of arbitrarily selected T cell receptor (TCR) beta-chain sequences. Healthy individuals displayed a highly diverse repertoire, with a median frequency of individual TCR beta-chain sequences of 1 in 2.4 x 10(7) CD4 T cells. In RA patients, the median TCR beta-chain frequency was increased 10-fold, indicating marked contraction of the repertoire (P < 0.001). The loss in TCR diversity was not limited to CD4 memory T cells but also involved the compartment of naive T cells, suggesting that it reflected an abnormality in T cell repertoire formation and not a consequence of antigen recognition in the synovium. Also, control patients with chronic inflammatory disease such as hepatitis C expressed a diverse repertoire indistinguishable from that of normals. Telomere length studies indicated an increased replicative history of peripheral CD4 T cells in RA patients, suggesting an enhanced turnover within the CD4 compartment. Compared with age-matched controls, terminal restriction fragment sizes were 1.7 kilobases shorter (P < 0.001). These data demonstrate an altered CD4 T cell homeostasis in RA that may contribute to the autoimmune response as well as to the immunodeficiency in these patients.

Crystals of the esterase EstB were obtained at 277 K with the hanging-drop technique from polyethylene glycol 4000 solutions containing 2-propanol at pH 7.5. The crystals belong to the trigonal space group P3(1)21 (or P3(2)21) with cell dimensions a = b = 82.9 and c = 193.4 A (at 100 K). The crystals diffract beyond a resolution of 2.0 A.

Crystals of the hydroxynitrile lyase from Hevea brasiliensis overexpressed in Pichia pastoris have been obtained by the hanging-drop technique at 294 K with ammonium sulfate and PEG 400 as precipitants. The crystals belong to the orthorhombic space group C222(1) with cell dimensions of a = 47.6, b = 106.8 and c = 128.2 A. The crystals diffract to about 2.5 A resolution on a rotating-anode X-ray source.

Crystal structure determinations of two orthorhombic (P2(1)2(1)2(1)) crystal modifications of the biliverdin apomyoglobin complex are described. The two structures were determined by X-ray diffraction at 100 K to a resolution of 1.5 A and 1.4 A. Both crystal forms were grown by hanging-drop techniques, using phosphate as precipitant. The structures were solved by molecular replacement and refined to final R-values of 19.4% and 21.2%. Both structures are very similar with respect to the binding site and the conformation of the biliverdin chromophore, which occurs in a (P) helical conformation. It is located within the heme pocket, very close in position and orientation to the heme binding site in myoglobin. Two water molecules not present in the crystal structure of myoglobin are sequestered within the heme pocket in the biliverdin-apomyoglobin complex, and they are engaged in hydrogen bonding to the biliverdin and to the protein. Comparison with structural results from an earlier NMR study of the same complex shows good agreement.

A crystal of the FabI protein from Escherichia coli has been obtained from polyethylene glycol (M(r) = 400) solution with sodium citrate at pH 8.5, by the hanging-drop technique at 4 degrees C. The crystal belongs to the hexagonal space group P6(1)22 (or P6(5)22) with cell dimensions of a = b = 81.1 A and c = 331.5 A. There are two molecules in the asymmetric unit and the crystal diffracts to 2.5 A resolution.

Crystals of a 40 kDa p-cresolyl-cobamide containing protein from Sporomusa ovata have been obtained from polyethyleneglycol solutions at pH 8.5 by the hanging drop technique. The crystals belong to space group C222(1) with cell dimensions a = 110.5(0.2) A, b = 144.0 (0.2) A, c = 110.4 (0.1) A. They diffract to 2.2 A resolution on a rotating anode X-ray source and are suitable for high resolution X-ray diffraction studies.

The three-dimensional X-ray structure of a recombinant human mitochondrial manganese superoxide dismutase (MnSOD) (chain length 198 residues) was determined by the method of molecular replacement using the related structure of MnSOD from Thermus thermophilus as a search model. This tetrameric human MnSOD crystallizes in space group P2(1)2(1)2 with a dimer in the asymmetric unit (Wagner, U.G., Werber, M.M., Beck, Y., Hartman, J.R., Frolow, F., & Sussman, J.L., 1989, J. Mol. Biol. 206, 787-788). Refinement of the protein structure (3,148 atoms with Mn and no solvents), with restraints maintaining noncrystallographic symmetry, converged at an R-factor of 0.207 using all data from 8.0 to 3.2 A resolution and group thermal parameters. The monomer-monomer interactions typical of bacterial Fe- and Mn-containing SODs are retained in the human enzyme, but the dimer-dimer interactions that form the tetramer are very different from those found in the structure of MnSOD from T. thermophilus. In human MnSOD one of the dimers is rotated by 84 degrees relative to its equivalent in the thermophile enzyme. As a result the monomers are arranged in an approximately tetrahedral array, the dimer-dimer packing is more intimate than observed in the bacterial MnSOD from T. thermophilus, and the dimers interdigitate. The metal-ligand interactions, determined by refinement and verified by computation of omit maps, are identical to those observed in T. thermophilus MnSOD.

The genetically engineered human manganese superoxide dismutase crystallizes in space group P2(1)2(1)2 with a = 75.51 A, b = 79.00 A, c = 67.95 A. At room temperature the crystals are not stable against radiation, so we cooled them to 90 K and collected a data set to 3 A resolution at this temperature.