Yasumitsu Sakamoto

The impact of a single-nucleotide mutation of bgl2 on cellulase induction in a Trichoderma reesei mutant

BackgroundThe filamentous fungus Trichoderma reesei (anamorph of Hypocrea jecorina) produces increased cellulase expression when grown on cellulose or its derivatives as a sole carbon source. It has been believed that β-glucosidases of T. reesei not only metabolize cellobiose but also contribute in the production of inducers of cellulase gene expression by their transglycosylation activity. The cellulase hyper-producing mutant PC-3-7 developed in Japan has enhanced cellulase production ability when cellobiose is used as the inducer. The comparative genomics analysis of PC-3-7 and its parent revealed a single-nucleotide mutation within the bgl2 gene encoding intracellular β-glucosidase II (BGLII/Cel1a), giving rise to an amino acid substitution in PC-3-7, which could potentially account for the enhanced cellulase expression when these strains are cultivated on cellulose and cellobiose.ResultsTo analyze the effects of the BGLII mutation in cellulase induction, we constructed both a bgl2 revertant and a disruptant. Enzymatic analysis of the transformant lysates showed that the strain expressing mutant BGLII exhibited weakened cellobiose hydrolytic activity, but produced some transglycosylation products, suggesting that the SNP in bgl2 strongly diminished cellobiase activity, but did not result in complete loss of function of BGLII. The analysis of the recombinant BGLII revealed that transglycosylation products might be oligosaccharides, composed probably of glucose linked β-1,4, β-1,3, or a mixture of both. PC-3-7 revertants of bgl2 exhibited reduced expression and inducibility of cellulase during growth on cellulose and cellobiose substrates. Furthermore, the effect of this bgl2 mutation was reproduced in the common strain QM9414 in which the transformants showed cellulase production comparable to that of PC-3-7.ConclusionWe conclude that BGLII plays an important role in cellulase induction in T. reesei and that the bgl2 mutation in PC-3-7 brought about enhanced cellulase expression on cellobiose. The results of the investigation using PC-3-7 suggested that other mutation(s) in PC-3-7 could also contribute to cellulase induction. Further investigation is essential to unravel the mechanism responsible for cellulase induction in T. reesei.

Identification of the Catalytic Triad of Family S46 Exopeptidases, Closely Related to Clan PA Endopeptidases

The exopeptidases of family S46 are exceptional, as the closest homologs of these enzymes are the endopeptidases of clan PA. The three-dimensional structure of S46 enzymes is unknown and only one of the catalytic residues, the serine, has been identified. The catalytic histidine and aspartate residues are not experimentally identified. Here we present phylogenetic and experimental data that identify all residues of the catalytic triad of S46 peptidase, dipeptidyl aminopeptidase BII (DAP BII) from Pseudoxanthomonas mexicana WO24. Phylogenetic comparison with the protein and S46 peptidases, revealed His-86, Ser-657, and five aspartate residues as possible catalytic residues. Mutation studies identified the catalytic triad of DAP BII as His-86, Asp-224, and Ser-657, while secondary structure analysis predicted an extended alpha-helical domain in between Asp-224 and Ser-657. This domain is unique for family S46 exopeptidases and its absence from the endopeptidases of clan PA might be key to their different hydrolysis activities.

S46 Peptidases are the First Exopeptidases to be Members of Clan PA

The dipeptidyl aminopeptidase BII (DAP BII) belongs to a serine peptidase family, S46. The amino acid sequence of the catalytic unit of DAP BII exhibits significant similarity to those of clan PA endopeptidases, such as chymotrypsin. However, the molecular mechanism of the exopeptidase activity of family S46 peptidase is unknown. Here, we report crystal structures of DAP BII. DAP BII contains a peptidase domain including a typical double β-barrel fold and previously unreported α-helical domain. The structures of peptide complexes revealed that the α-helical domain covers the active-site cleft and the side chain of Asn330 in the domain forms hydrogen bonds with the N-terminus of the bound peptide. These observations indicate that the α-helical domain regulates the exopeptidase activity of DAP BII. Because S46 peptidases are not found in mammals, we expect that our study will be useful for the design of specific inhibitors of S46 peptidases from pathogens.

Structural and mutational analyses of dipeptidyl peptidase 11 from Porphyromonas gingivalis reveal the molecular basis for strict substrate specificity

The dipeptidyl peptidase 11 from Porphyromonas gingivalis (PgDPP11) belongs to the S46 family of serine peptidases and preferentially cleaves substrates with Asp/Glu at the P1 position. The molecular mechanism underlying the substrate specificity of PgDPP11, however, is unknown. Here, we report the crystal structure of PgDPP11. The enzyme contains a catalytic domain with a typical double β-barrel fold and a recently identified regulatory α-helical domain. Crystal structure analyses, docking studies, and biochemical studies revealed that the side chain of Arg673 in the S1 subsite is essential for recognition of the Asp/Glu side chain at the P1 position of the bound substrate. Because S46 peptidases are not found in mammals and the Arg673 is conserved among DPP11s, we anticipate that DPP11s could be utilised as targets for antibiotics. In addition, the present structure analyses could be useful templates for the design of specific inhibitors of DPP11s from pathogenic organisms.

Crystallization and preliminary X-ray crystallographic studies of dipeptidyl aminopeptidase BII from Pseudoxanthomonas mexicana WO24

Dipeptidyl aminopeptidase BII from Pseudoxanthomonas mexicana WO24 (DAP BII) is able to cleave a variety of dipeptides from the amino-terminus of substrate peptides. For crystallographic studies, DAP BII was overproduced in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data to 2.3 Å resolution were collected using an orthorhombic crystal form belonging to space group P2(1)2(1)2(1), with unit-cell parameters a = 76.55, b = 130.86, c = 170.87 Å. Structural analysis by the multi-wavelength anomalous diffraction method is in progress.

Crystallization and Preliminary X-Ray Crystallographic Analysis of Plasmodium Falciparum S-Adenosyl-L-Homocysteine Hydrolase

S-adenosyl-l-homocysteine hydrolase from a malaria parasite Plasmodium falciparum (PfSAHH) has been crystallized by the vapor diffusion method. The crystals belong to an orthorhombic space group P212121 with the cell dimensions of a = 76.66 A, b = 86.31 A, and c = 335.6 A. There are four subunits (one tetramer) per asymmetric unit. X-ray diffraction data have been collected up to 2.8 A resolution. Self-rotation function studies suggest that the tetrameric PfSAHH molecule has the 222 point group symmetry.

Role of α/β interface in F1 ATPase rotational catalysis probed by inhibitors and mutations

The F1 sector of ATP synthase (FOF1) synthesizes or hydrolyses ATP via a rotational catalysis mechanism that couples chemical reaction with subunit rotation. Phytopolyphenols such as curcumin can inhibit bulk phase F1 ATPase activity by extending the catalytic dwell time during subunit rotation (Sekiya, M., Hisasaka, R., Iwamoto-Kihara, A., Futai, M., Nakanishi-Matsui, M., Biochem. Biophys. Res. Commun. 452 (2014) 940-944). Citreoviridin, a polyene α-pyrone mycotoxin isolated from Penicillium sp, also inhibits ATPase activity. Molecular docking and mutational analysis indicated that these compounds interact with a region near the β-subunit Arg398 residue that lies at the interface with the α-subunit. Binding of these inhibitors lowered the rotation rate and increased the duration of the catalytic dwell synergistically with substitution of β-subunit Ser174 to Phe (βS174F), which rendered the enzyme defective for conformational transmission between β-subunits of different catalytic stages. Furthermore, substitution of α-subunit Glu402 to Ala (αE402A) in the α/β-interface also decreased the rotation rate by increasing the duration of the catalytic dwell. Interestingly, this mutation restored the catalytic dwell of the βS174F variant to that of the wild-type enzyme. These results suggest that the α/β-interface is involved in conformational changes of the β-subunit during rotational catalysis.

JAK3 inhibitor VI is a mutant specific inhibitor for epidermal growth factor receptor with the gatekeeper mutation T790M

AIM To identify non-quinazoline kinase inhibitors effective against drug resistant mutants of epidermal growth factor receptor (EGFR). METHODS A kinase inhibitor library was subjected to screening for specific inhibition pertaining to the in vitro kinase activation of EGFR with the gatekeeper mutation T790M, which is resistant to small molecular weight tyrosine kinase inhibitors (TKIs) for EGFR in non-small cell lung cancers (NSCLCs). This inhibitory effect was confirmed by measuring autophosphorylation of EGFR T790M/L858R in NCI-H1975 cells, an NSCLC cell line harboring the gatekeeper mutation. The effects of a candidate compound, Janus kinase 3 (JAK3) inhibitor VI, on cell proliferation were evaluated using the MTT assay and were compared between T790M-positive and -negative lung cancer cell lines. JAK3 inhibitor VI was modeled into the ATP-binding pocket of EGFR T790M/L858R. Potential physical interactions between the compound and kinase domains of wild-type (WT) or mutant EGFRs or JAK3 were estimated by calculating binding energy. The gatekeeper residues of EGFRs and JAKs were aligned to discuss the similarities among EGFR T790M and JAKs. RESULTS We found that JAK3 inhibitor VI, a known inhibitor for JAK3 tyrosine kinase, selectively inhibits EGFR T790M/L858R, but has weaker inhibitory effects on the WT EGFR in vitro. JAK3 inhibitor VI also specifically reduced autophosphorylation of EGFR T790M/L858R in NCI-H1975 cells upon EGF stimulation, but did not show the inhibitory effect on WT EGFR in A431 cells. Furthermore, JAK3 inhibitor VI suppressed the proliferation of NCI-H1975 cells, but showed limited inhibitory effects on the WT EGFR-expressing cell lines A431 and A549. A docking simulation between JAK3 inhibitor VI and the ATP-binding pocket of EGFR T790M/L858R predicted a potential binding status with hydrogen bonds. Estimated binding energy of JAK3 inhibitor VI to EGFR T790M/L858R was more stable than its binding energy to the WT EGFR. Amino acid sequence alignments revealed that the gatekeeper residues of JAK family kinases are methionine in WT, similar to EGFR T790M, suggesting that TKIs for JAKs may also be effective for EGFR T790M. CONCLUSION Our findings demonstrate that JAK3 inhibitor VI is a gatekeeper mutant selective TKI and offer a strategy to search for new EGFR T790M inhibitors.

Crystallization and preliminary X‐ray crystallographic analysis of human autotaxin

Autotaxin (ATX), which is also known as ectonucleotide pyrophosphatase/phosphodiesterase 2 (NPP2 or ENPP2) or phosphodiesterase Iα (PD-Iα), is an extracellular lysophospholipase D which generates lysophosphatidic acid (LPA) from lysophosphatidylcholine (LPC). ATX stimulates tumour-cell migration, angiogenesis and metastasis and is an attractive target for cancer therapy. For crystallographic studies, the α isoform of human ATX was overproduced in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 3.0 Å resolution from a monoclinic crystal form belonging to space group C2, with unit-cell parameters a = 311.4, b = 147.9, c = 176.9 Å, β = 122.6°.

Crystal Structure analyses of dipeptidyl aminopeptidase BII fromP. mexicanaWO24.

The peptidase family S46 that contains the dipeptidyl aminopeptidase BII (DAP BII) from Pseudoxanthomonas mexicana WO24 is the only exopeptidase family in clan PA peptidases. Our present phylogenetic and experimental studies indicated that the catalytic triad of DAP BII is composed of His 86, Asp 224 and Ser 657 and implied that unknown large helical domains involved in exopeptidase activity[1]. However, three-dimensional structure of a family S46 peptidase has not yet been reported. Thus, the crystal structure of DAP BII is essential not only to understand the catalytic mechanism of family S46 peptidases but also to clarify the structural origin of the exo-type peptidase activities of these enzymes. Recently, we have successfully crystallized the DAP BII and collected X-ray diffraction data to 2.3 Å resolution from the crystal. This crystal belonging to space group P212121, with unit-cell parameters a = 76.55 Å, b = 130.86 Å, c= 170.87 Å[2]. Structural analysis by the multi-wavelength anomalous diffraction method is underway[3]. Here, we report the first crystallization and structural analysis of the DAP BII from P. mexicana WO24 as family S46 peptidase. Other enzymes that belong to this family are DPP7 and DPP11 from Porphyromonas gingivalis, DPP11 from Porphyromonas endodontalis (periodontal pathogen) and DPP11 from Shewanella putrefaciens (multidrug resistance associated opportunistic pathogen). These gram-negative bacterial pathogens are known to asaccharolytic. Especially, Porphyromonas gingivalis is known to utilize dipeptides, instead of free amino acids, as energy source and cellular material. Since S46 peptidases are not found in mammals, we expect our study will be useful for the discovery of specific inhibitors to S46 peptidases from these pathogens.