Sine Larsen

Stimulation of lignocellulosic biomass hydrolysis by proteins of glycoside hydrolase family 61: structure and function of a large, enigmatic family.

Currently, the relatively high cost of enzymes such as glycoside hydrolases that catalyze cellulose hydrolysis represents a barrier to commercialization of a biorefinery capable of producing renewable transportable fuels such as ethanol from abundant lignocellulosic biomass. Among the many families of glycoside hydrolases that catalyze cellulose and hemicellulose hydrolysis, few are more enigmatic than family 61 (GH61), originally classified based on measurement of very weak endo-1,4-beta-d-glucanase activity in one family member. Here we show that certain GH61 proteins lack measurable hydrolytic activity by themselves but in the presence of various divalent metal ions can significantly reduce the total protein loading required to hydrolyze lignocellulosic biomass. We also solved the structure of one highly active GH61 protein and find that it is devoid of conserved, closely juxtaposed acidic side chains that could serve as general proton donor and nucleophile/base in a canonical hydrolytic reaction, and we conclude that the GH61 proteins are unlikely to be glycoside hydrolases. Structure-based mutagenesis shows the importance of several conserved residues for GH61 function. By incorporating the gene for one GH61 protein into a commercial Trichoderma reesei strain producing high levels of cellulolytic enzymes, we are able to reduce by 2-fold the total protein loading (and hence the cost) required to hydrolyze lignocellulosic biomass.

Structure of the conserved domain of ANAC, a member of the NAC family of transcription factors.

The structure of the DNA‐binding NAC domain of Arabidopsis ANAC (abscisic‐acid‐responsive NAC) has been determined by X‐ray crystallography to 1.9 Å resolution (Protein Data Bank codes 1UT4 and 1UT7). This is the first structure determined for a member of the NAC family of plant‐specific transcriptional regulators. NAC proteins are characterized by their conserved N‐terminal NAC domains that can bind both DNA and other proteins. NAC proteins are involved in developmental processes, including formation of the shoot apical meristem, floral organs and lateral shoots, as well as in plant hormonal control and defence. The NAC domain does not possess a classical helix–turn–helix motif; instead it reveals a new transcription factor fold consisting of a twisted β‐sheet surrounded by a few helical elements. The functional dimer formed by the NAC domain was identified in the structure, which will serve as a structural template for understanding NAC protein function at the molecular level.

Rhamnogalacturonan acetylesterase elucidates the structure and function of a new family of hydrolases

BACKGROUND The complex polysaccharide rhamnogalacturonan constitutes a major part of the hairy region of pectin. It can have different types of carbohydrate sidechains attached to the rhamnose residues in the backbone of alternating rhamnose and galacturonic acid residues; the galacturonic acid residues can be methylated or acetylated. Aspergillus aculeatus produces enzymes that are able to perform a synergistic degradation of rhamnogalacturonan. The deacetylation of the backbone by rhamnogalacturonan acetylesterase (RGAE) is an essential prerequisite for the subsequent action of the enzymes that cleave the glycosidic bonds. RESULTS The structure of RGAE has been determined at 1.55 A resolution. RGAE folds into an alpha/beta/alpha structure. The active site of RGAE is an open cleft containing a serine-histidine-aspartic acid catalytic triad. The position of the three residues relative to the central parallel beta sheet and the lack of the nucleophilic elbow motif found in structures possessing the alpha/beta hydrolase fold show that RGAE does not belong to the alpha/beta hydrolase family. CONCLUSIONS Structural and sequence comparisons have revealed that, despite very low sequence similarities, RGAE is related to seven other proteins. They are all members of a new hydrolase family, the SGNH-hydrolase family, which includes the carbohydrate esterase family 12 as a distinct subfamily. The SGNH-hydrolase family is characterised by having four conserved blocks of residues, each with one completely conserved residue; serine, glycine, asparagine and histidine, respectively. Each of the four residues plays a role in the catalytic function.

Structural and functional insight into how the Plasmodium falciparum VAR2CSA protein mediates binding to chondroitin sulfate A in placental malaria.

Background: VAR2CSA expressing Plasmodium falciparum parasites cause placental malaria by interacting with chondroitin sulfate A (CSA) on placental syncytiotrophoblasts. Results: The CSA-binding site in VAR2CSA lies within the N-terminal DBL2X domain, which maps to the center of the compact VAR2CSA structure. Conclusion: VAR2CSA fragments based on the CSA-binding region are potent vaccine candidates. Significance: The data presented has important implications for vaccine development. Malaria is a major global health problem. Pregnant women are susceptible to infection regardless of previously acquired immunity. Placental malaria is caused by parasites capable of sequestering in the placenta. This is mediated by VAR2CSA, a parasite antigen that interacts with chondroitin sulfate A (CSA). One vaccine strategy is to block this interaction with VAR2CSA-specific antibodies. It is a priority to define a small VAR2CSA fragment that can be used in an adhesion blocking vaccine. In this, the obvious approach is to define regions of VAR2CSA involved in receptor binding. It has been shown that full-length recombinant VAR2CSA binds specifically to CSA with nanomolar affinity, and that the CSA-binding site lies in the N-terminal part of the protein. In this study we define the minimal binding region by truncating VAR2CSA and analyzing CSA binding using biosensor technology. We show that the core CSA-binding site lies within the DBL2X domain and parts of the flanking interdomain regions. This is in contrast to the idea that single domains do not possess the structural requirements for specific CSA binding. Small-angle x-ray scattering measurements enabled modeling of VAR2CSA and showed that the CSA-binding DBL2X domain is situated in the center of the structure. Mutating classic sulfate-binding sites in VAR2CSA, along with testing dependence of ionic interactions, suggest that the CSA binding is not solely dependent on the sulfated CSA structure. Based on these novel PfEMP1 structure-function studies, we have constructed a small VAR2CSA antigen that has the capacity to induce highly adhesion-blocking antibodies.

Structures of β-Ketoacyl-Acyl Carrier Protein Synthase I Complexed with Fatty Acids Elucidate its Catalytic Machinery

BACKGROUND beta-ketoacyl-acyl carrier protein synthase (KAS) I is vital for the construction of the unsaturated fatty acid carbon skeletons characterizing E. coli membrane lipids. The new carbon-carbon bonds are created by KAS I in a Claisen condensation performed in a three-step enzymatic reaction. KAS I belongs to the thiolase fold enzymes, of which structures are known for five other enzymes. RESULTS Structures of the catalytic Cys-Ser KAS I mutant with covalently bound C10 and C12 acyl substrates have been determined to 2.40 and 1.85 A resolution, respectively. The KAS I dimer is not changed by the formation of the complexes but reveals an asymmetric binding of the two substrates bound to the dimer. A detailed model is proposed for the catalysis of KAS I. Of the two histidines required for decarboxylation, one donates a hydrogen bond to the malonyl thioester oxo group, and the other abstracts a proton from the leaving group. CONCLUSIONS The same mechanism is proposed for KAS II, which also has a Cys-His-His active site triad. Comparison to the active site architectures of other thiolase fold enzymes carrying out a decarboxylation step suggests that chalcone synthase and KAS III with Cys-His-Asn triads use another mechanism in which both the histidine and the asparagine interact with the thioester oxo group. The acyl binding pockets of KAS I and KAS II are so similar that they alone cannot provide the basis for their differences in substrate specificity.

Estimated H-atom anisotropic displacement parameters: a comparison between different methods and with neutron diffraction results

Anisotropic displacement parameters (ADPs) are compared for H atoms estimated using three recently described procedures, both among themselves and with neutron diffraction results. The results convincingly demonstrate that all methods are capable of giving excellent results for several benchmark systems and identify systematic discrepancies for several atom types. A revised and extended library of internal H-atom mean-square displacements is presented for use with Madsens SHADE web server [J. Appl. Cryst. (2006), 39, 757-758; http://shade.ki.ku.dk], and the improvement over the original SHADE results is substantial, suggesting that this is now the most readily and widely applicable of the three approximate procedures. Using this new library--SHADE2--it is shown that, in line with expectations, a segmented rigid-body description of the heavy atoms yields only a small improvement in the agreement with neutron results. The SHADE2 library, now incorporated in the SHADE web server, is recommended as a routine procedure for deriving estimates of H-atom ADPs suitable for use in charge-density studies on molecular crystals, and its widespread use should reveal remaining deficiencies and perhaps overcome the inherent bias in the majority of such studies.

The crystal structure of the flavin containing enzyme dihydroorotate dehydrogenase A from Lactococcus lactis

BACKGROUND . Dihydroorotate dehydrogenase (DHOD) is a flavin mononucleotide containing enzyme, which catalyzes the oxidation of (S)-dihydroorotate to orotate, the fourth step in the de novo biosynthesis of pyrimidine nucleotides. Lactococcus lactis contains two genes encoding different functional DHODs whose sequences are only 30% identical. One of these enzymes, DHODA, is a highly efficient dimer, while the other, DHODB, shows optimal activity only in the presence of an iron-sulphur cluster containing protein with which it forms a complex tetramer. Sequence alignments have identified three different families among the DHODs: the two L. lactis enzymes belong to two of the families, whereas the enzyme from E. coli is a representative of the third. As no three-dimensional structures of DHODs are currently available, we set out to determine the crystal structure of DHODA from L. lactis. The differences between the two L. lactis enzymes make them particularly interesting for studying flavoprotein redox reactions and for identifying the differences between the enzyme families. RESULTS . The crystal structure of DHODA has been determined to 2.0 resolution. The enzyme is a dimer of two crystallographically independent molecules related by a non-crystallographic twofold axis. The protein folds into and alpha/beta barrel with the flavin molecule sitting between the top of the barrel and a subdomain formed by several barrel inserts. Above the flavin isoalloxazine ring there is a small water filled cavity, completely buried beneath the protein surface and surrounded by many conserved residues. This cavity is proposed as the substrate-binding site. CONCLUSIONS . The crystal structure has allowed the function of many of the conserved residues in DHODs to be identified: many of these are associated with binding the flavin group. Important differences were identified in some of the active-site residues which vary across the distinct DHOD families, implying significant mechanistic differences. The substrate cavity, although buried, is located beneath a highly conserved loop which is much less ordered than the rest of the protein and may be important in giving access to the cavity. The location of the conserved residues surrounding this cavity suggests the potential orientation of the substrate.

Three-dimensional structure of a recombinant peroxidase from Coprinus cinereus at 2.6 Å resolution

The structure of a recombinant peroxidase from the ink cap, Coprinus cinereus, CiP, is reported to 2.6 Å resolution and refined to a R‐value of 18.1%. The structure was solved by molecular replacement using the coordinates from a newly published ligninase structure, LiP. CiP crystallizes in space group P212121) with two independent molecules in the asymmetric unit related by the vector 0.29b + 0.5C. The two CiP molecules are structurally identical; each contains two Ca2+ ions in positions equivalent to those found in the LiP structure. Two N‐acetylglucosamines and one mannose residue were fitted into the density adjacent to two of the three predicted glycosylation sites. The refinement also included 40 and 41 water molecules, respectively, in the two CiP molecules. The structure of CiP displays a folding very similar to that of LiP. The active sites are almost identical in the LiP and CiP structures. CiP has a much larger opening to the active site than LiP.

Structural basis for the function of Bacillus subtilis phosphoribosyl-pyrophosphate synthetase.

Here we report the first three-dimensional structure of a phosphoribosylpyrophosphate (PRPP) synthetase. PRPP is an essential intermediate in several biosynthetic pathways. Structures of the Bacillus subtilis PRPP synthetase in complex with analogs of the activator phosphate and the allosteric inhibitor ADP show that the functional form of the enzyme is a hexamer. The individual subunits fold into two domains, both of which resemble the type I phosphoribosyltransfereases. The active site is located between the two domains and includes residues from two subunits. Phosphate and ADP bind to the same regulatory site consisting of residues from three subunits of the hexamer. In addition to identifying residues important for binding substrates and effectors, the structures suggest a novel mode of allosteric regulation.

The 1.62 Å structure of Thermoascus aurantiacus endoglucanase: completing the structural picture of subfamilies in glycoside hydrolase family 5

The crystal structure of Thermoascus aurantiacus endoglucanase (Cel5A), a family 5 glycoside hydrolase, has been determined to 1.62 Å resolution by multiple isomorphous replacement with anomalous scattering. It is the first report of a structure in the subfamily to which Cel5A belongs. Cel5A consists solely of a catalytic module with compact eight‐fold β/α barrel architecture. The length of the tryptophan‐rich substrate binding groove suggests the presence of substrate binding subsites −4 to +3. Structural comparison shows that two glycines are completely conserved in the family, in addition to the two catalytic glutamates and six other conserved residues previously identified. Gly 44 in particular is part of a type IV C‐terminal helix capping motif, whose disruption is likely to affect the position of an essential conserved arginine. One aromatic residue (Trp 170 in Cel5A), not conserved in term of sequence, is nonetheless spatially conserved in the substrate binding groove. Its role might be to force the bend that occurs in the polysaccharide chain on binding, thus favoring substrate distortion at subsite −1.