Highlighting the factors governing transglycosylation in the GH5_5 endo-1,4-ß-glucanase RBcel1.

Transglycosylating glycoside hydrolases (GHs) offer great potential for the enzymatic synthesis of oligosaccharides. Although knowledge is progressing, there is no unique strategy to improve the transglycosylation yield. Obtaining efficient enzymatic tools for glycan synthesis with GHs remains dependent on an improved understanding of the molecular factors governing the balance between hydrolysis and transglycosylation. This enzymatic and structural study of RBcel1, a transglycosylase from the GH5_5 subfamily isolated from an uncultured bacterium, aims to unravel such factors. The size of the acceptor and donor sugars was found to be critical since transglycosylation is efficient with oligosaccharides at least the size of cellotetraose as the donor and cellotriose as the acceptor. The reaction pH is important in driving the balance between hydrolysis and transglycosylation: hydrolysis is favored at pH values below 8, while transglycosylation becomes the major reaction at basic pH. Solving the structures of two RBcel1 variants, RBcel1_E135Q and RBcel1_Y201F, in complex with ligands has brought to light some of the molecular factors behind transglycosylation. The structure of RBcel1_E135Q in complex with cellotriose allowed a +3 subsite to be defined, in accordance with the requirement for cellotriose as a transglycosylation acceptor. The structure of RBcel1_Y201F has been obtained with several transglycosylation intermediates, providing crystallographic evidence of transglycosylation. The catalytic cleft is filled with (i) donors ranging from cellotriose to cellohexaose in the negative subsites and (ii) cellobiose and cellotriose in the positive subsites. Such a structure is particularly relevant since it is the first structure of a GH5 enzyme in complex with transglycosylation products that has been obtained with neither of the catalytic glutamate residues modified.

Glycoside hydrolase family 5: structural snapshots highlighting the involvement of two conserved residues in catalysis.

The ability of retaining glycoside hydrolases (GHs) to transglycosylate is inherent to the double-displacement mechanism. Studying reaction intermediates, such as the glycosyl-enzyme intermediate (GEI) and the Michaelis complex, could provide valuable information to better understand the molecular factors governing the catalytic mechanism. Here, the GEI structure of RBcel1, an endo-1,4-ß-glucanase of the GH5 family endowed with transglycosylase activity, is reported. It is the first structure of a GH5 enzyme covalently bound to a natural oligosaccharide with the two catalytic glutamate residues present. The structure of the variant RBcel1_E135A in complex with cellotriose is also reported, allowing a description of the entire binding cleft of RBcel1. Taken together, the structures deliver different snapshots of the double-displacement mechanism. The structural analysis revealed a significant movement of the nucleophilic glutamate residue during the reaction. Enzymatic assays indicated that, as expected, the acid/base glutamate residue is crucial for the glycosylation step and partly contributes to deglycosylation. Moreover, a conserved tyrosine residue in the -1 subsite, Tyr201, plays a determinant role in both the glycosylation and deglycosylation steps, since the GEI was trapped in the RBcel1_Y201F variant. The approach used to obtain the GEI presented here could easily be transposed to other retaining GHs in clan GH-A.

M42 aminopeptidase catalytic site: the structural and functional role of a strictly conserved aspartate residue.

The M42 aminopeptidases are a family of dinuclear aminopeptidases widely distributed in Prokaryotes. They are potentially associated to the proteasome, achieving complete peptide destruction. Their most peculiar characteristic is their quaternary structure, a tetrahedron-shaped particle made of twelve subunits. The catalytic site of M42 aminopeptidases is defined by seven conserved residues. Five of them are involved in metal ion binding which is important to maintain both the activity and the oligomeric state. The sixth conserved residue, a glutamate, is the catalytic base deprotonating the water molecule during peptide bond hydrolysis. The seventh residue is an aspartate whose function remains poorly understood. This aspartate residue, however, must have a critical role as it is strictly conserved in all MH clan enzymes. It forms some kind of catalytic triad with the histidine residue and the metal ion of the M2 binding site. We assess its role in TmPep1050, an M42 aminopeptidase of Thermotoga maritima, through a mutational approach. Asp-62 was substituted with alanine, asparagine, or glutamate residue. The Asp-62 substitutions completely abolished TmPep1050 activity and impeded dodecamer formation. They also interfered with metal ion binding as only one cobalt ion is bound per subunit instead of two. The structure of Asp62Ala variant was solved at 1.5 Å showing how the substitution has an impact on the active site fold. We propose a structural role for Asp-62, helping to stabilize a crucial loop in the active site and to position correctly the catalytic base and a metal ion ligand of the M1 site.

X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050.

The M42 aminopeptidases form functionally active complexes made of 12 subunits. Their assembly process appears to be regulated by their metal ion cofactors triggering a dimer-dodecamer transition. Upon metal ion binding, several structural modifications occur in the active site and at the interaction interface, shaping dimers to promote the self-assembly. To observe such modifications, stable oligomers must be isolated prior to structural study. Reported here is a method that allows the purification of stable dodecamers and dimers of TmPep1050, an M42 aminopeptidase of T. maritima, and their structure determination by X-ray crystallography. Dimers were prepared from dodecamers by removing metal ions with a chelating agent. Without their cofactor, dodecamers became less stable and were fully dissociated upon heating. The oligomeric structures were solved by the straightforward molecular replacement approach. To illustrate the methodology, the structure of a TmPep1050 variant, totally impaired in metal ion binding, is presented showing no further breakdown of dimers to monomers.

How metal cofactors drive dimer-dodecamer transition of the M42 aminopeptidase TmPep1050 of Thermotoga maritima.

The M42 aminopeptidases are dinuclear aminopeptidases displaying a peculiar tetrahedron-shaped structure with 12 subunits. Their quaternary structure results from the self-assembly of six dimers controlled by their divalent metal ion cofactors. The oligomeric-state transition remains debated despite the structural characterization of several archaeal M42 aminopeptidases. The main bottleneck is the lack of dimer structures, hindering the understanding of structural changes occurring during the oligomerization process. We present the first dimer structure of an M42 aminopeptidase, TmPep1050 of Thermotoga maritima, along with the dodecamer structure. The comparison of both structures has allowed us to describe how the metal ion cofactors modulate the active-site fold and, subsequently, affect the interaction interface between dimers. A mutational study shows that the M1 site strictly controls dodecamer formation. The dodecamer structure of TmPep1050 also reveals that a part of the dimerization domain delimits the catalytic pocket and could participate in substrate binding.

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.

Identifying reaction modules in metabolic pathways: bioinformatic deduction and experimental validation of a new putative route in purine catabolism.

BACKGROUND: Enzymes belonging to mechanistically diverse superfamilies often display similar catalytic mechanisms. We previously observed such an association in the case of the cyclic amidohydrolase superfamily whose members play a role in related steps of purine and pyrimidine metabolic pathways. To establish a possible link between enzyme homology and chemical similarity, we investigated further the neighbouring steps in the respective pathways. RESULTS: We identified that successive reactions of the purine and pyrimidine pathways display similar chemistry. These mechanistically-related reactions are often catalyzed by homologous enzymes. Detection of series of similar catalysis made by succeeding enzyme families suggested some modularity in the architecture of the central metabolism. Accordingly, we introduce the concept of a reaction module to define at least two successive steps catalyzed by homologous enzymes in pathways alignable by similar chemical reactions. Applying such a concept allowed us to propose new function for misannotated paralogues. In particular, we discovered a putative ureidoglycine carbamoyltransferase (UGTCase) activity. Finally, we present experimental data supporting the conclusion that this UGTCase is likely to be involved in a new route in purine catabolism. CONCLUSIONS: Using the reaction module concept should be of great value. It will help us to trace how the primordial promiscuous enzymes were assembled progressively in functional modules, as the present pathways diverged from ancestral pathways to give birth to the present-day mechanistically diversified superfamilies. In addition, the concept allows the determination of the actual function of misannotated proteins.

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.

Processing of HEBP1 by cathepsin D gives rise to F2L, the agonist of formyl peptide receptor 3.

The peptide F2L was previously characterized as a high-affinity natural agonist for the human formyl peptide receptor (FPR) 3. F2L is an acetylated 21-aa peptide corresponding with the N terminus of the intracellular heme-binding protein 1 (HEBP1). In the current work, we have investigated which proteases were able to generate the F2L peptide from its precursor HEBP1. Structure-function analysis of F2L identified three amino acids, G(3), N(7), and S(8), as the most important for interaction of the peptide with FPR3. We expressed a C-terminally His-tagged form of human HEBP1 in yeast and purified it to homogeneity. The purified protein was used as substrate to identify proteases generating bioactive peptides for FPR3-expressing cells. A conditioned medium from human monocyte-derived macrophages was able to generate bioactivity from HEBP1, and this activity was inhibited by pepstatin A. Cathepsin D was characterized as the protease responsible for HEBP1 processing, and the bioactive product was identified as F2L. We have therefore determined how F2L, the specific agonist of FPR3, is generated from the intracellular protein HEBP1, although it is unknown in which compartment the processing by cathepsin D occurs in vivo.

Selection systems based on dominant-negative transcription factors for precise genetic engineering.

Diverse tools are available for performing genetic modifications of microorganisms. However, new methods still need to be developed for performing precise genomic engineering without introducing any undesirable side-alteration. Indeed for functional analyses of genomic elements, as well as for some industrial applications, only the desired mutation should be introduced at the locus considered. This article describes a new approach fulfilling these requirements, based on the use of selection systems consisting in truncated genes encoding dominant-negative transcription factors. We have demonstrated dominant-negative effects mediated by truncated Gal4p and Arg81p proteins in Saccharomyces cerevisiae, interfering with galactose and arginine metabolic pathways, respectively. These genes can be used as positive and negative markers, since they provoke both growth inhibition on substrates and resistance to specific drugs. These selection markers have been successfully used for precisely deleting HO and URA3 in wild yeasts. This genetic engineering approach could be extended to other microorganisms.

Overexpression, physicochemical characterization, and modeling of a hyperthermophilic pyrococcus furiosus type 2 IPP isomerase.

In the first step of this study, type 2 isopentenyl diphosphate isomerase (IDI2) from Pyrococcus furiosus (pf-IDI2), a hyperthermophilic microorganism, was cloned and overexpressed in E. coli. After purification, hyperthermophilic behavior of this protein was approached by means of enzymatic assays and thermal denaturation studies. Compared with the mesophilic Streptococcus pneumoniae IDI2, which unfolds and looses activity above 50 degrees C, pf-IDI2 is still folded and active at 80 degrees C. Molecular modeling was applied, in a parallel step, to understand the molecular basis of thermal stability. Comparison of IDI2 from S. pneumoniae, T. thermophilus, and P. furiosus suggested that additional charged residues present in the hyperthermophilic enzyme might contribute to its higher thermal stability. This could increase the number of salt bridges between monomers of IDI2 in P. furiosus enzyme and, hence, decrease flexibility of loops or N-terminal segment, thereby enhancing its thermal stability.