Biochemical, Bioinformatic, and Structural Comparisons of Transketolases and Position of Human Transketolase in the Enzyme Evolution.

Transketolases (TKs) are key enzymes of the pentose phosphate pathway, regulating several other critical pathways in cells. Considering their metabolic importance, TKs are expected to be conserved throughout evolution. However, Tittmann et al. (J Biol Chem, 2010, 285(41): 31559-31570) demonstrated that Homo sapiens TK (hsTK) possesses several structural and kinetic differences compared to bacterial TKs. Here, we study 14 TKs from pathogenic bacteria, fungi, and parasites and compare them with hsTK using biochemical, bioinformatic, and structural approaches. For this purpose, six new TK structures are solved by X-ray crystallography, including the TK of Plasmodium falciparum. All of these TKs have the same general fold as bacterial TKs. This comparative study shows that hsTK greatly differs from TKs from pathogens in terms of enzymatic activity, spatial positions of the active site, and monomer-monomer interface residues. An ubiquitous structural pattern is identified in all TKs as a six-residue histidyl crown around the TK cofactor (thiamine pyrophosphate), except for hsTK containing only five residues in the crown. Residue mapping of the monomer-monomer interface and the active site reveals that hsTK contains more unique residues than other TKs. From an evolutionary standpoint, TKs from animals (including H. sapiens) and Schistosoma sp. belong to a distinct structural group from TKs of bacteria, plants, fungi, and parasites, mostly based on a different linker between domains, raising hypotheses regarding evolution and regulation.

Structural and Functional Characterization of Drosophila melanogaster α-Amylase.

Insects rely on carbohydrates such as starch and glycogen as an energy supply for growth of larvae and for longevity. In this sense α-amylases have essential roles under extreme conditions, e.g., during nutritional or temperature stress, thereby contributing to survival of the insect. This makes them interesting targets for combating insect pests. Drosophila melanogaster α-amylase, DMA, which belongs to the glycoside hydrolase family 13, sub family 15, has been studied from an evolutionary, biochemical, and structural point of view. Our studies revealed that the DMA enzyme is active over a broad temperature and pH range, which is in agreement with the fluctuating environmental changes with which the insect is confronted. Crystal structures disclosed a new nearly fully solvated metal ion, only coordinated to the protein via Gln263. This residue is only conserved in the subgroup of D. melanogaster and may thus contribute to the enzyme adaptive response to large temperature variations. Studies of the effect of plant inhibitors and the pseudo-tetrasaccharide inhibitor acarbose on DMA activity, allowed us to underline the important role of the so-called flexible loop on activity/inhibition, but also to suggest that the inhibition modes of the wheat inhibitors WI-1 and WI-3 on DMA, are likely different.

The Candida glabrata glycogen branching enzyme structure reveals unique features of branching enzymes of the Saccharomycetaceae phylum.

Branching enzymes (BE) are responsible for the formation of branching points at the 1,6 position in glycogen and starch, by catalyzing the cleavage of α-1,4-linkages and the subsequent transfer by introducing α-1,6-linked glucose branched points. BEs are found in the large GH13 family, eukaryotic BEs being mainly classified in the GH13_8 subfamily, GH13_9 grouping almost exclusively prokaryotic enzymes. With the aim of contributing to the understanding of the mode of recognition and action of the enzymes belonging to GH13_8, and to the understanding of features distinguishing these enzymes from those belonging to subfamily 13_9, we solved the crystal structure of the glycogen branching enzyme (GBE) from the yeast Candida glabrata, CgGBE, in ligand-free forms and in complex with a maltotriose. The structures revealed the presence of a domain already observed in Homo sapiens and Oryza sativa BEs that we named α-helical N-terminal domain, in addition to the three conserved domains found in BE. We confirmed by phylogenetic analysis that this α-helical N-terminal domain is always present in the GH13_8 enzymes suggesting that it could actually present a signature for this subfamily. We identified two binding sites in the α-helical N-terminal domain and in the carbohydrate binding module 48 (CBM48), respectively, which show a unique structural organization only present in the Saccharomycotina phylum. Our structural and phylogenetic investigation provides new insight into the structural characterization of GH13_8 GBE revealing that unique structural features only present in the Saccharomycotina phylum thereby conferring original properties to this group of enzymes.

Exploring molecular determinants of polysaccharide lyase family 6-1 enzyme activity.

The polysaccharide lyase family 6 (PL6) represents one of the 41 polysaccharide lyase families classified in the CAZy database with the vast majority of its members being alginate lyases grouped into three subfamilies, PL6_1-3. To decipher the mode of recognition and action of the enzymes belonging to subfamily PL6_1, we solved the crystal structures of Pedsa0632, Patl3640, Pedsa3628 and Pedsa3807, which all show different substrate specificities and mode of action (endo-/exolyase). Thorough exploration of the structures of Pedsa0632 and Patl3640 in complex with their substrates as well as docking experiments confirms that the conserved residues in subsites -1 to +3 of the catalytic site form a common platform that can accommodate various types of alginate in a very similar manner but with a series of original adaptations bringing them their specificities of action. From comparative studies with existing structures of PL6_1 alginate lyases, we observe that in the right-handed parallel ß-helix fold shared by all these enzymes, the substrate-binding site harbors the same overall conserved structures and organization. Despite this apparent similarity, it appears that members of the PL6_1 subfamily specifically accommodate and catalyze the degradation of different alginates suggesting that this common platform is actually a highly adaptable and specific tool.

Helices on Interdomain Interface Couple Catalysis in the ATPPase Domain with Allostery in Plasmodium falciparum GMP Synthetase.

GMP synthetase catalyses the conversion of XMP to GMP through a series of reactions that include hydrolysis of Gln to generate ammonia in the glutamine amidotransferase (GATase) domain, activation of XMP to adenyl-XMP intermediate in the ATP pyrophosphatase (ATPPase) domain and reaction of ammonia with the intermediate to generate GMP. The functioning of GMP synthetases entails bidirectional domain crosstalk, which leads to allosteric activation of the GATase domain, synchronization of catalytic events and tunnelling of ammonia. Herein, we have taken recourse to the analysis of structures of GMP synthetases, site-directed mutagenesis and steady-state and transient kinetics on the Plasmodium falciparum enzyme to decipher the molecular basis of catalysis in the ATPPase domain and domain crosstalk. Our results suggest an arrangement at the interdomain interface, of helices with residues that play roles in ATPPase catalysis as well as domain crosstalk enabling the coupling of ATPPase catalysis with GATase activation. Overall, the study enhances our understanding of GMP synthetases, which are drug targets in many infectious pathogens.

Structural determinants increasing flexibility confer cold adaptation in psychrophilic phosphoglycerate kinase.

Crystal structures of phosphoglycerate kinase (PGK) from the psychrophile Pseudomonas sp. TACII 18 have been determined at high resolution by X-ray crystallography methods and compared with mesophilic, thermophilic and hyperthermophilic counterparts. PGK is a two-domain enzyme undergoing large domain movements to catalyze the production of ATP from 1,3-biphosphoglycerate and ADP. Whereas the conformational dynamics sustaining the catalytic mechanism of this hinge-bending enzyme now seems rather clear, the determinants which underlie high catalytic efficiency at low temperatures of this psychrophilic PGK were unknown. The comparison of the three-dimensional structures shows that multiple (global and local) specific adaptations have been brought about by this enzyme. Together, these reside in an overall increased flexibility of the cold-adapted PGK thereby allowing a better accessibility to the active site, but also a potentially more disordered transition state of the psychrophilic enzyme, due to the destabilization of some catalytic residues.

Two different centered monoclinic crystals of the E. coli outer-membrane protein OmpF originate from the same building block.

Macromolecule crystal formation can be divided in two major steps: 1. the formation of a nucleus and 2. the growth of this nucleus into a full mature crystal. The latter is well described and understood, while the former remains elusive due to the difficulty to study it and is described by nucleation theories. Here we report the structure of the Escherichia coli outer membrane porin OmpF in two centered monoclinic space groups. Strikingly, the two crystals originate from the same building block, made of two trimers of OmpF interacting via their rough side. The different crystallization conditions trigger the formation of distinct arrangement of these building blocks, leading to the formation of translational non-crystallographic symmetry (tNCS) in one case, made possible by the loose lateral packing mediated by detergents. In light of nucleation theories, these results allow us to speculate that these two crystals originate from nuclei made of either clusters of building blocks, or already forming columns that later associate laterally using detergents as glue.

The attractive recombinant phytase from Bacillus licheniformis: biochemical and molecular characterization.

The phyL gene encoding phytase from the industrial strain Bacillus licheniformis ATCC 14580 (PhyL) was cloned, sequenced, and overexpressed in Escherichia coli. Biochemical characterization demonstrated that the recombinant enzyme has an apparent molecular weight of nearly 42 kDa. Interestingly, this enzyme was optimally active at 70-75 °C and pH 6.5-7.0. This enzyme is distinguishable by the fact that it preserved more than 40 % of its activity at wide range of temperatures from 4 to 85 °C. This new phytase displayed also a high specific activity of 316 U/mg. For its maximal activity and thermostability, this biocatalyst required only 0.6 mM of Ca(2+) ion and exhibited high catalytic efficiency of 8.3 s(-1) µM(-1) towards phytic acid.

Carving out a Glycoside Hydrolase Active Site for Incorporation into a New Protein Scaffold Using Deep Network Hallucination.

Enzymes are indispensable biocatalysts for numerous industrial applications, yet stability, selectivity, and restricted substrate recognition present limitations for their use. Despite the importance of enzyme engineering in overcoming these limitations, success is often challenged by the intricate architecture of enzymes derived from natural sources. Recent advances in computational methods have enabled the de novo design of simplified scaffolds with specific functional sites. Such scaffolds may be advantageous as platforms for enzyme engineering. Here, we present a strategy for the de novo design of a simplified scaffold of an endo-α-N-acetylgalactosaminidase active site, a glycoside hydrolase from the GH101 enzyme family. Using a combination of trRosetta hallucination, iterative cycles of deep-learning-based structure prediction, and ProteinMPNN sequence design, we designed proteins with 290 amino acids incorporating the active site while reducing the molecular weight by over 100 kDa compared to the initial endo-α-N-acetylgalactosaminidase. Of 11 tested designs, six were expressed as soluble monomers, displaying similar or increased thermostabilities compared to the natural enzyme. Despite lacking detectable enzymatic activity, the experimentally determined crystal structures of a representative design closely matched the design with a root-mean-square deviation of 1.0 Å, with most catalytically important side chains within 2.0 Å. The results highlight the potential of scaffold hallucination in designing proteins that may serve as a foundation for subsequent enzyme engineering.

Mutations inducing an active-site aperture in Rhizobium sp. sucrose isomerase confer hydrolytic activity.

Sucrose isomerase is an enzyme that catalyzes the production of sucrose isomers of high biotechnological and pharmaceutical interest. Owing to the complexity of the chemical synthesis of these isomers, isomaltulose and trehalulose, enzymatic conversion remains the preferred method for obtaining these products. Depending on the microbial source, the ratio of the sucrose-isomer products varies significantly. In studies aimed at understanding and explaining the underlying molecular mechanisms of these reactions, mutations obtained using a random-mutagenesis approach displayed a major hydrolytic activity. Two of these variants, R284C and F164L, of sucrose isomerase from Rhizobium sp. were therefore crystallized and their crystal structures were determined. The three-dimensional structures of these mutants allowed the identification of the molecular determinants that favour hydrolytic activity compared with transferase activity. Substantial conformational changes resulting in an active-site opening were observed, as were changes in the pattern of water molecules bordering the active-site region.

Identification of critical residues for the activity and thermostability of Streptomyces sp. SK glucose isomerase.

The role of residue 219 in the physicochemical properties of D-glucose isomerase from Streptomyces sp. SK strain (SKGI) was investigated by site-directed mutagenesis and structural studies. Mutants G219A, G219N, and G219F were generated and characterized. Comparative studies of their physicochemical properties with those of the wild-type enzyme highlighted that mutant G219A displayed increased specific activity and thermal stability compared to that of the wild-type enzyme, while for G219N and G219F, these properties were considerably decreased. A double mutant, SKGI F53L/G219A, displayed a higher optimal temperature and a higher catalytic efficiency than both the G219A mutant and the wild-type enzyme and showed a half-life time of about 150 min at 85 °C as compared to 50 min for wild-type SKGI. Crystal structures of SKGI wild-type and G219A enzymes were solved to 1.73 and 2.15 Å, respectively, and showed that the polypeptide chain folds into two structural domains. The larger domain consists of a (ß/α)8 unit, and the smaller domain forms a loop of α helices. Detailed analyses of the three-dimensional structures highlighted minor but important changes in the active site region as compared to that of the wild-type enzyme leading to a displacement of both metal ions, and in particular that in site M2. The structural analyses moreover revealed how the substitution of G219 by an alanine plays a crucial role in improving the thermostability of the mutant enzyme.

The acid tolerant and cold-active ß-galactosidase from Lactococcus lactis strain is an attractive biocatalyst for lactose hydrolysis.

The gene encoding the ß-galactosidase from the dairy Lactococcus lactis IL1403 strain was cloned, sequenced and overexpressed in Escherichia coli. The purified enzyme has a tetrameric arrangement composed of four identical 120 kDa subunits. Biochemical characterization showed that it is optimally active within a wide range of temperatures from 15 to 55 °C and of pH from 6.0 to 7.5. For its maximal activity this enzyme requires only 0.8 mM Fe(2+) and 1.6 mM Mg(2+). Purified protein displayed a high catalytic efficiency of 102 s(-1) mM(-1) for lactose. The enzyme stability was increased by immobilization mainly at low pH (from 4.0 to 5.5) and high temperatures (55 and 60 °C). The bioconversion of lactose using the L. lactis ß-galactosidase allows the production of lactose with a high bioconversion rate (98 %) within a wide range of pH and temperature.

GMP Synthetase: Allostery, Structure, and Function.

Glutamine amidotransferases (GATs) catalyze the hydrolysis of glutamine and transfer the generated ammonia to diverse metabolites. The two catalytic activities, glutaminolysis and the subsequent amination of the acceptor substrate, happen in two distinct catalytic pockets connected by a channel that facilitates the movement of ammonia. The de novo pathway for the synthesis of guanosine monophosphate (GMP) from xanthosine monophosphate (XMP) is enabled by the GAT GMP synthetase (GMPS). In most available crystal structures of GATs, the ammonia channel is evident in their native state or upon ligand binding, providing molecular details of the conduit. In addition, conformational changes that enable the coordination of the two catalytic chemistries are also informed by the available structures. In contrast, despite the first structure of a GMPS being published in 1996, the understanding of catalysis in the acceptor domain and inter-domain crosstalk became possible only after the structure of a glutamine-bound mutant of Plasmodium falciparum GMPS was determined. In this review, we present the current status of our understanding of the molecular basis of catalysis in GMPS, becoming the first comprehensive assessment of the biochemical function of this intriguing enzyme.

Purine containing carbonucleoside phosphonate analogues as novel chemotype for Plasmodium falciparum Inhibition.

The nucleotidase ISN1 is a potential therapeutic target of the purine salvage pathway of the malaria parasite Plasmodium falciparum. We identified PfISN1 ligands by in silico screening of a small library of nucleos(t)ide analogues and by thermal shift assays. Starting from a racemic cyclopentyl carbocyclic phosphonate scaffold, we explored the diversity on the nucleobase moiety and also proposed a convenient synthetic pathway to access the pure enantiomers of our initial hit (compound (±)-2). 2,6-Disubstituted purine containing derivatives such as compounds 1, (±)-7e and ß-L-(+)-2 showed the most potent inhibition of the parasite in vitro, with low micromolar IC50 values. These results are remarkable considering the anionic nature of nucleotide analogues, which are known to lack activity in cell culture experiments due to their scarce capacity to cross cell membranes. For the first time, we report the antimalarial activity of a carbocyclic methylphosphonate nucleoside with an L-like configuration.

Engineered glucose isomerase from Streptomyces sp. SK is resistant to Ca²âº inhibition and Co²âº independent.

The role of two amino acid residues linked to the two catalytic histidines His54 and His220 in kinetics and physicochemical properties of the Streptomyces sp. SK glucose isomerase (SKGI) was investigated by site-directed mutagenesis and molecular modeling. Two single mutations, F53L and G219D, and a double mutation F53L/G219D was introduced into the xylA SKGI gene. The F53L mutation increases the thermostability and the catalytic efficiency and also slightly shifts the optimum pH from 6.5 to 7, but displays a profile being similar to that of the wild-type enzyme concerning the effect of various metal ions. The G219D mutant is resistant to calcium inhibition retaining about 80% of its residual activity in 10 mM Ca²âº instead of 10% for the wild-type. This variant is activated by Mn²âº ions, but not Co²âº, as seen for the wild-type enzyme. It does not require the latter for its thermostability, but has its half-life time displaced from 50 to 20 min at 85°C. The double mutation F53L/G219D restores the thermostability as seen for the wild-type enzyme while maintaining the resistance to the calcium inhibition. Molecular modeling suggests that the increase in thermostability is due to new hydrophobic interactions stabilizing α2 helix and that the resistance to calcium inhibition is a result of narrowing the binding site of catalytic ion.

Structural and molecular determinants of Candida glabrata metacaspase maturation and activation by calcium.

Metacaspases are caspase-like homologs which undergo a complex maturation process involving multiple intra-chain cleavages resulting in a composite enzyme made of a p10 and a p20 domain. Their proteolytic activity involving a cysteine-histidine catalytic dyad, show peptide bond cleavage specificity in the C-terminal to lysine and arginine, with both maturation- and catalytic processes being calcium-dependent. Here, we present the structure of a metacaspase from the yeast Candida glabrata, CgMCA-I, in complex with a unique calcium along with a structure in which three magnesium ions are bound. We show that the Ca2+ ion interacts with a loop in the vicinity of the catalytic site. The reorganization of this cation binding loop, by bringing together the two catalytic residues, could be one of the main structural determinants triggering metacaspase activation. Enzymatic exploration of CgMCA-I confirmed that the maturation process implies a trans mechanism with sequential cleavages.

Tertiary and Quaternary Structure Organization in GMP Synthetases: Implications for Catalysis.

Glutamine amidotransferases, enzymes that transfer nitrogen from Gln to various cellular metabolites, are modular, with the amidotransferase (GATase) domain hydrolyzing Gln, generating ammonia and the acceptor domain catalyzing the addition of nitrogen onto its cognate substrate. GMP synthetase (GMPS), an enzyme in the de novo purine nucleotide biosynthetic pathway, is a glutamine amidotransferase that catalyzes the synthesis of GMP from XMP. The reaction involves activation of XMP though adenylation by ATP in the ATP pyrophosphatase (ATPPase) active site, followed by channeling and attack of NH3 generated in the GATase pocket. This complex chemistry entails co-ordination of activity across the active sites, allosteric activation of the GATase domain to modulate Gln hydrolysis and channeling of ammonia from the GATase to the acceptor active site. Functional GMPS dimers associate through the dimerization domain. The crystal structure of the Gln-bound complex of Plasmodium falciparum GMPS (PfGMPS) for the first time revealed large-scale domain rotation to be associated with catalysis and leading to the juxtaposition of two otherwise spatially distal cysteinyl (C113/C337) residues. In this manuscript, we report on an unusual structural variation in the crystal structure of the C89A/C113A PfGMPS double mutant, wherein a larger degree of domain rotation has led to the dissociation of the dimeric structure. Furthermore, we report a hitherto overlooked signature motif tightly related to catalysis.

Dynamics of the secreted frizzled related protein Sizzled and potential implications for binding to bone morphogenetic protein-1 (BMP-1).

Sizzled (Szl) is both a secreted frizzled related protein (sFRP) and a naturally occurring inhibitor of the zinc metalloproteinase bone morphogenetic protein-1 (BMP-1), a key regulator of extracellular matrix assembly and growth factor activation. Here we present a new crystal structure for Szl which differs from that previously reported by a large scale (90°) hinge rotation between its cysteine-rich and netrin-like domains. We also present results of a molecular docking analysis showing interactions likely to be involved in the inhibition of BMP-1 activity by Szl. When compared with known structures of BMP-1 in complex with small molecule inhibitors, this reveals features that may be helpful in the design of new inhibitors to prevent the excessive accumulation of extracellular matrix that is the hallmark of fibrotic diseases.

The acid-tolerant L-arabinose isomerase from the mesophilic Shewanella sp. ANA-3 is highly active at low temperatures.

BACKGROUND: L-arabinose isomerases catalyse the isomerization of L-arabinose into L-ribulose at insight biological systems. At industrial scale of this enzyme is used for the bioconversion of D-galactose into D-tagatose which has many applications in pharmaceutical and agro-food industries. The isomerization reaction is thermodynamically equilibrated, and therefore the bioconversion rates is shifted towards tagatose when the temperature is increased. Moreover, to prevent secondary reactions it will be of interest to operate at low pH. The profitability of this D-tagatose production process is mainly related to the use of lactose as cheaper raw material. In many dairy products it will be interesting to produce D-tagatose during storage. This requires an efficient L-arabinose isomerase acting at low temperature and pH values. RESULTS: The gene encoding the L-arabinose isomerase from Shewanella sp. ANA-3 was cloned and overexpressed in Escherichia coli. The purified protein has a tetrameric arrangement composed by four identical 55 kDa subunits. The biochemical characterization of this enzyme showed that it was distinguishable by its maximal activity at low temperatures comprised between 15-35°C. Interestingly, this biocatalyst preserves more than 85% of its activity in a broad range of temperatures from 4.0 to 45°C. Shewanella sp. ANA-3 L-arabinose isomerase was also optimally active at pH 5.5-6.5 and maintained over 80% of its activity at large pH values from 4.0 to 8.5. Furthermore, this enzyme exhibited a weak requirement for metallic ions for its activity evaluated at 0.6 mM Mn2+. Stability studies showed that this protein is highly stable mainly at low temperature and pH values. Remarkably, T268K mutation clearly enhances the enzyme stability at low pH values. Use of this L-arabinose isomerase for D-tagatose production allows the achievement of attractive bioconversion rates of 16% at 4°C and 34% at 35°C. CONCLUSIONS: Here we reported the purification and the biochemical characterization of the novel Shewanella sp. ANA-3 L-arabinose isomerase. Determination of the biochemical properties demonstrated that this enzyme was highly active at low temperatures. The generated T268K mutant displays an increase of the enzyme stability essentially at low pH. These features seem to be very attractive for the bioconversion of D-galactose into D-tagatose at low temperature which is very interesting from industrial point of view.

C-terminal residues regulate localization and function of the antiapoptotic protein Bfl-1.

Unlike other antiapoptotic members of the Bcl-2 family, Bfl-1 does not contain a well defined C-terminal transmembrane domain, and whether the C-terminal tail of Bfl-1 functions as a membrane anchor is not yet clearly established. The molecular modeling study of the full-length Bfl-1 performed within this work suggests that Bfl-1 may co-exist in two distinct conformational states: one in which its C-terminal helix alpha9 is inserted in the hydrophobic groove formed by the BH1-3 domains of Bfl-1 and one with its C terminus. Parallel analysis of the subcellular localization of Bfl-1 indicates that even if Bfl-1 may co-exist in two distinct conformational states, most of the endogenous protein is tightly associated with the mitochondria by its C terminus in both healthy and apoptotic peripheral blood lymphocytes as well as in malignant B cell lines. However, the helix alpha9 of Bfl-1, and therefore the binding of Bfl-1 to mitochondria, is not absolutely required for the antiapoptotic activity of Bfl-1. A particular feature of Bfl-1 is the amphipathic character of its C-terminal helix alpha9. Our data clearly indicate that this property of helix alpha9 is required for the anchorage of Bfl-1 to the mitochondria but also regulates the antiapoptotic function Bfl-1.