Crystal structure determination of Pseudomonas stutzeri A1501 endoglucanase Cel5A: the search for a molecular basis for glycosynthesis in GH5_5 enzymes.

The discovery of new glycoside hydrolases that can be utilized in the chemoenzymatic synthesis of carbohydrates has emerged as a promising approach for various biotechnological processes. In this study, recombinant Ps_Cel5A from Pseudomonas stutzeri A1501, a novel member of the GH5_5 subfamily, was expressed, purified and crystallized. Preliminary experiments confirmed the ability of Ps_Cel5A to catalyze transglycosylation with cellotriose as a substrate. The crystal structure revealed several structural determinants in and around the positive subsites, providing a molecular basis for a better understanding of the mechanisms that promote and favour synthesis rather than hydrolysis. In the positive subsites, two nonconserved positively charged residues (Arg178 and Lys216) were found to interact with cellobiose. This adaptation has also been reported for transglycosylating ß-mannanases of the GH5_7 subfamily.

High yield production in seven days of Coriolopsis gallica 1184 laccase at 50 L scale; enzyme purification and molecular characterization.

The optimization of culture conditions for high yield laccase production by white rot fungi has been extensively studied. However, to achieve short time laccase production remains a major challenge in several cases. The present study investigated an optimal process for production of Coriolopsis gallica 1184 laccase in a high yield of 200 900 Ul(-1) in 7 d by 50 L scale submerged fermentation. Coriolopsis gallica 1184 laccase appeared as a robust enzyme against downstream process; only 13.5 % of laccase activity was lost at the end of downstream procedure. The pure enzyme appeared as a one-species laccase, with a molecular mass of 66 kDa as determined by SDS-PAGE. The pH optimum for 2,2'-azino-bis-[3-ethyltiazoline-6-sulfonate] oxidation ranged between 2.5 and 3.0 in 100 mM tartrate buffer. Optimum temperature for laccase activity was determined to be around 70 °C. The kinetic of laccase was investigated with four phenolic substrates. The lowest Km values (17 and 20 µM) were found for ABTS and guaiacol, respectively. Coriolopsis gallica 1184 laccase was characterized by mass spectrometry and shows that C. gallica 1184_LacI is very likely a new member of the AA1_1 subfamily. Our results clearly show high competitive potential of the robust extracellular C. gallica 1184 laccase to use it in different industrial processes.

Three-dimensional structure of RBcel1, a metagenome-derived psychrotolerant family GH5 endoglucanase.

RBcel1 is an endoglucanase belonging to glycoside hydrolase family 5 subfamily 5 (GH5_5) that was recently identified from a soil metagenome library from the Antarctic. Unlike its closest structural homologue (Cel5A from Thermoascus aurantiacus), this enzyme was reported to be able to catalyze transglycosylation reactions and has putatively been implicated in the bacterial cellulose-synthesis process. Here, the structure of RBcel1 at 1.4 Å resolution, solved by molecular replacement, is reported. The structure and putative substrate-binding site are described and compared with those of other GH5_5 subfamily members.

Functional characterization of two M42 aminopeptidases erroneously annotated as cellulases.

Several aminopeptidases of the M42 family have been described as tetrahedral-shaped dodecameric (TET) aminopeptidases. A current hypothesis suggests that these enzymes are involved, along with the tricorn peptidase, in degrading peptides produced by the proteasome. Yet the M42 family remains ill defined, as some members have been annotated as cellulases because of their homology with CelM, formerly described as an endoglucanase of Clostridium thermocellum. Here we describe the catalytic functions and substrate profiles CelM and of TmPep1050, the latter having been annotated as an endoglucanase of Thermotoga maritima. Both enzymes were shown to catalyze hydrolysis of nonpolar aliphatic L-amino acid-pNA substrates, the L-leucine derivative appearing as the best substrate. No significant endoglucanase activity was measured, either for TmPep1050 or CelM. Addition of cobalt ions enhanced the activity of both enzymes significantly, while both the chelating agent EDTA and bestatin, a specific inhibitor of metalloaminopeptidases, proved inhibitory. Our results strongly suggest that one should avoid annotating members of the M42 aminopeptidase family as cellulases. In an updated assessment of the distribution of M42 aminopeptidases, we found TET aminopeptidases to be distributed widely amongst archaea and bacteria. We additionally observed that several phyla lack both TET and tricorn. This suggests that other complexes may act downstream from the proteasome.

The CphAII protein from Aquifex aeolicus exhibits a metal-dependent phosphodiesterase activity.

The CphAII protein from the hyperthermophile Aquifex aeolicus shows the five conserved motifs of the metallo-ß-lactamase (MBL) superfamily and presents 28% identity with the Aeromonas hydrophila subclass B2 CphA MBL. The gene encoding CphAII was amplified by PCR from the A. aeolicus genomic DNA and overexpressed in Escherichia coli using a pLex-based expression system. The recombinant CphAII protein was purified by a combination of heating (to denature E. coli proteins) and two steps of immobilized metal affinity chromatography. The purified enzyme preparation did not exhibit a ß-lactamase activity but showed a metal-dependent phosphodiesterase activity versus bis-p-nitrophenyl phosphate and thymidine 5'-monophosphate p-nitrophenyl ester, with an optimum at 85°C. The circular dichroism spectrum was in agreement with the percentage of secondary structures characteristic of the MBL αßßα fold.

Three factors that modulate the activity of class D ß-lactamases and interfere with the post-translational carboxylation of Lys70.

The activity of class D ß-lactamases is dependent on Lys70 carboxylation in the active site. Structural, kinetic and affinity studies show that this post-translational modification can be affected by the presence of a poor substrate such as moxalactam but also by the V117T substitution. Val117 is a strictly conserved hydrophobic residue located in the active site. In addition, inhibition of class D ß-lactamases by chloride ions is due to a competition between the side chain carboxylate of the modified Lys70 and chloride ions. Determination of the individual kinetic constants shows that the deacylation of the acyl-enzyme is the rate-limiting step for the wild-type OXA-10 ß-lactamase.

Metal-induced conformational changes in ZneB suggest an active role of membrane fusion proteins in efflux resistance systems.

Resistance nodulation cell division (RND)-based efflux complexes mediate multidrug and heavy-metal resistance in many Gram-negative bacteria. Efflux of toxic compounds is driven by membrane proton/substrate antiporters (RND protein) in the plasma membrane, linked by a membrane fusion protein (MFP) to an outer-membrane protein. The three-component complex forms an efflux system that spans the entire cell envelope. The MFP is required for the assembly of this complex and is proposed to play an important active role in substrate efflux. To better understand the role of MFPs in RND-driven efflux systems, we chose ZneB, the MFP component of the ZneCAB heavy-metal efflux system from Cupriavidus metallidurans CH34. ZneB is shown to be highly specific for Zn(2+) alone. The crystal structure of ZneB to 2.8 A resolution defines the basis for metal ion binding in the coordination site at a flexible interface between the beta-barrel and membrane proximal domains. The conformational differences observed between the crystal structures of metal-bound and apo forms are monitored in solution by spectroscopy and chromatography. The structural rearrangements between the two states suggest an active role in substrate efflux through metal binding and release.

Crystal structure of the human monoacylglycerol lipase, a key actor in endocannabinoid signaling.

2-Arachidonoylglycerol plays a major role in endocannabinoid signaling, and is tightly regulated by the monoacylglycerol lipase (MAGL). Here we report the crystal structure of human MAGL. The protein crystallizes as a dimer, and despite structural homologies to haloperoxidases and esterases, it distinguishes itself by a wide and hydrophobic access to the catalytic site. An apolar helix covering the active site also gives structural insight into the amphitropic character of MAGL, and likely explains how MAGL interacts with membranes to recruit its substrate. Docking of 2-arachidonoylglycerol highlights a hydrophobic and a hydrophilic cavity that accommodate the lipid into the catalytic site. Moreover, we identified Cys201 as the crucial residue in MAGL inhibition by N-arachidonylmaleimide, a sulfhydryl-reactive compound. Beside the advance in the knowledge of endocannabinoids degradation routes, the structure of MAGL paves the way for future medicinal chemistry works aimed at the design of new drugs exploiting 2-arachidonoylglycerol transmission.

AdcAII, a new pneumococcal Zn-binding protein homologous with ABC transporters: biochemical and structural analysis.

Regulation of metal homeostasis is vital for pathogenic bacteria facing drastic metal concentration changes in various locations within the host during invasion. Metal-binding receptors (MBRs), one of the extracellular components of ATP-binding cassette transporters, have been shown to be essential in this process. Streptococcus pneumoniae expresses two characterized MBRs: PsaA and AdcA, two extracellular lipoproteins encoded by the psaABCD and adcRCBA operons, respectively. The Mn- and Zn-uptake functions of PsaA and AdcA, respectively, have been well established. Here we describe AdcAII as a third putative S. pneumoniae MBR. The analysis of a phylogenetic tree built from the sequence alignment of 68 proteins reveals a subgroup of members displaying an unusual genetic operon organisation. The adcAII gene belongs to a 6670-nucleotide-long transcript spanning the spr0903 to spr0907 loci encoding for the CcdA, thioredoxine, YfnA, AdcAII and PhtD proteins. Two adjacent repeats of imperfect AdcR-binding consensus sequence were identified upstream of the adcAII gene, suggesting a transcriptional co-regulation of adcAII and phtD genes. Biophysical and structural studies of recombinant AdcAII were performed to identify the metal specificity of the protein. Using electrospray mass spectrometry in native conditions, we found that Zn was bound to recombinant AdcAII. Screening of the effect of 10 cationic ions on the thermal stability of AdcAII revealed that Zn had the most pronounced stabilizing effect. The crystal structure of AdcAII has been solved to 2.4 A resolution. One Zn ion is bound to each AdcAII molecule in a symmetrical active site composed of three His and one Glu. The structure almost perfectly superimposed on the known MBR structures. The presence of a flexible 15-residue-long loop close to the metal-binding site is specific to those specialized in Zn transport. Taken together, these functional and structural data provide new perspectives related to the physiological role of AdcAII in pneumococcus Zn homeostasis.

Crystal structure of the cold-active aminopeptidase from Colwellia psychrerythraea, a close structural homologue of the human bifunctional leukotriene A4 hydrolase.

The crystal structure of a cold-active aminopeptidase (ColAP) from Colwellia psychrerythraea strain 34H has been determined, extending the number of crystal structures of the M1 metallopeptidase family to four among the 436 members currently identified. In agreement with their sequence similarity, the overall structure of ColAP displayed a high correspondence with leukotriene A4 hydrolase (LTA4H), a human bifunctional enzyme that converts leukotriene A4 (LTA4) in the potent chemoattractant leukotriene B4. Indeed, both enzymes are composed of three domains, an N-terminal saddle-like domain, a catalytic thermolysin-like domain, and a less conserved C-terminal alpha-helical flat spiral domain. Together, these domains form a deep cavity harboring the zinc binding site formed by residues included in the conserved HEXXHX(18)H motif. A detailed structural comparison of these enzymes revealed several plausible determinants of ColAP cold adaptation. The main differences involve specific amino acid substitutions, loop content and solvent exposure, complexity and distribution of ion pairs, and differential domain flexibilities. Such elements may act synergistically to allow conformational flexibility needed for an efficient catalysis in cold environments. Furthermore, the region of ColAP corresponding to the aminopeptidase active site of LTA4H is much more conserved than the suggested LTA4 substrate binding region. This observation supports the hypothesis that this region of the LTA4H active site has evolved in order to fit the lipidic substrate.

Structure of the plasmid-mediated class C beta-lactamase ACT-1.

The crystallographic structure of ACT-1, which is the first plasmid-mediated AmpC-type beta-lactamase to have been completely analyzed in terms of nucleotide sequence and which has a high degree of sequence similarity to the chromosomal AmpC enzymes of Enterobacter cloacae and the plasmid-encoded MIR-1, has been solved at 2.4 A resolution. The overall structure of ACT-1 is similar to those of other class C beta-lactamases, such as the AmpC enzymes from E. cloacae P99 and Escherichia coli.

Disulfiram is an inhibitor of human purified monoacylglycerol lipase, the enzyme regulating 2-arachidonoylglycerol signaling.

Monoacylglycerol lipase (MAGL) is a key enzyme responsible for the termination of endocannabinoid signaling. Its crucial role in 2-arachidonoylglycerol (2-AG) metabolism, together with the numerous pharmacological properties mediated by this endocannabinoid, emphasize the interest in MAGL as therapeutic target, along with the need to design potent and selective inhibitors. Meanwhile, the complexity of 2-AG degradation pathways underscores the need to use a purified source of enzyme in evaluation studies of new inhibitors. We report here the first heterologous expression and purification of human MAGL. A highly pure protein was obtained and allowed us to measure the affinity of several MAGL inhibitors for the human enzyme. Importantly, disulfiram (tetraethylthiuram disulfide), a compound used to treat alcoholism, and other disulfide-containing compounds were shown to inhibit MAGL with good potency, likely through an interaction with cysteine residues.

Characterisation of KLUA-9, a beta-lactamase from extended-spectrum cephalosporin-susceptible Kluyvera ascorbata, and genetic organisation of bla(KLUA-9).

This study characterised the genetic environment of the chromosomally encoded bla(KLUA-9) gene from a clinical Kluyvera ascorbata isolate and performed a kinetic characterisation of KLUA-9. Purified KLUA-9 showed the highest catalytic efficacies towards benzylpenicillin, ampicillin, piperacillin, first-generation cephalosporins, cefuroxime and cefoperazone; like other 'cefotaximases', it showed a much higher rate of hydrolysis of cefotaxime than ceftazidime, whilst dicloxacillin, cefoxitin and imipenem behaved as poor substrates. A 9kb insert from K. ascorbata was cloned (Escherichia coli KK68C1) and sequenced. bla(KLUA-9) and its 266bp upstream flanking region (almost identical to the integron-associated bla(CTX-M-2)) are preceded by an aspat variant, a ypdABC-like operon and two open reading frames with unknown functions. Unlike ISCR1-associated bla(CTX-M-2) genes, we failed to detect the putative orf513 recombination sites. Instead, we were able to localise the 5bp target sites for insertion of ISEcp1B, suggesting that this element could be responsible for future (or still undetected) mobilisation of bla(KLUA-9) to more efficiently transferred elements.

Kinetic properties of four plasmid-mediated AmpC beta-lactamases.

The heterologous production in Escherichia coli, the purification, and the kinetic characterization of four plasmid-encoded class C beta-lactamases (ACT-1, MIR-1, CMY-2, and CMY-1) were performed. Except for their instability, these enzymes are very similar to the known chromosomally encoded AmpC beta-lactamases. Their kinetic parameters did not show major differences from those obtained for the corresponding chromosomal enzymes. However, the K(m) values of CMY-2 for cefuroxime, cefotaxime, and oxacillin were significantly decreased compared to those of the chromosomal AmpC enzymes. Finally, the susceptibility patterns of different E. coli hosts producing a plasmid- or a chromosome-encoded class C enzyme toward beta-lactam antibiotics are mainly due to the overproduction of the beta-lactamase in the periplasmic space of the bacteria rather than to a specific catalytic profile of the plasmid-encoded beta-lactamases.

The ybxI gene of Bacillus subtilis 168 encodes a class D beta-lactamase of low activity.

The ybxI gene of Bacillus subtilis 168 encodes a preprotein of 267 amino acid residues, including a putative signal peptide of 23 residues. The YbxI primary structure exhibits high similarity scores with two members of the superfamily of the serine penicillin-recognizing enzymes: the class D beta-lactamases and the hydrophilic carboxy-terminal domains of the BlaR and MecR penicillin receptors. To determine the function and the activity of this putative penicillin-recognizing enzyme, we have subcloned the ybxI gene in the pET-26b expression vector. Transformation of Escherichia coli BL21(DE3) by the recombinant plasmid pCIP51 resulted in the export of the mature YbxI in the periplasm as a water-soluble protein. The recombinant protein was purified to 95% homogeneity. YbxI interacts with several beta-lactam antibiotics and can hydrolyze some of them. YbxI is not inactivated by clavulanic acid. The YbxI function and its enzymatic activity in B. subtilis remain unknown. The acyl-enzyme obtained after incubation of YbxI with a fluorescent derivative of ampicillin can be detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, confirming that YbxI can be acylated by beta-lactam antibiotics. YbxI does not hydrolyze some of the standard substrates of D-alanyl-D-alanine peptidases, the targets of penicillin. YbxI belongs to the penicillin-recognizing enzyme family but has an activity intermediate between those of a penicillin-binding protein and a beta-lactamase.

A detailed kinetic study of Mox-1, a plasmid-encoded class C beta-lactamase.

Surveys of beta-lactamases in different parts of the world show an important increase in class C beta-lactamases, thus the study of these enzymes is becoming an important issue. We created an overproduction system for Mox-1, a plasmid class C beta-lactamase, by cloning the gene encoding this enzyme, and placing it under the control of a T7 promoter, using vector pET 28a. The enzyme, purified by ion exchange chromatography, was used to obtain the molecular mass (38246), the N-terminal sequence (GEASPVDPLRPVV), and pI (8.9), and to perform a detailed kinetic study. Cephalotin was used as reporter substrate in the case of poor substrates. The kinetic study showed that benzylpenicillin, cephalotin, cefcapene and moxalactam were good substrates for Mox-1 (k(cat)/K(m) values >2.5 x 10(6) M(-1) s(-1)). On the other hand, ceftazidime and cefepime were poor substrates for this enzyme (K(m) values >200 microM). Clavulanic acid had no inhibitory effect on Mox-1 (K(m)=30.2 mM), however aztreonam behaved as an inhibitor of Mox-1 (K(i)=2.85 microM).