Hiroshi Sakamoto

Structural basis for recognition of the tra mRNA precursor by the Sex-lethal protein

The Sex-lethal (Sxl) protein of Drosophila melanogaster regulates alternative splicing of the transformer (tra) messenger RNA precursor by binding to the tra polypyrimidine tract during the sex-determination process. The crystal structure has now been determined at 2.6u2009Å resolution of the complex formed between two tandemly arranged RNA-binding domains of the Sxl protein and a 12-nucleotide, single-stranded RNA derived from the tra polypyrimidine tract. The two RNA-binding domains have their β-sheet platforms facing each other to form a V-shaped cleft. The RNA is characteristically extended and bound in this cleft, where the UGUUUUUUU sequence is specifically recognized by the protein. This structure offers the first insight, to our knowledge, into how a protein binds specifically to a cognate RNA without any intramolecular base-pairing.

A fully linear-time approximation algorithm for grammar-based compression

Abstract A linear-time approximation algorithm for the grammar-based compression is presented. This is an optimization problem to minimize the size of a context-free grammar deriving a given string. For each string of length n , the algorithm guarantees O ( log n g ∗ ) approximation ratio without suffix tree construction.

Towards systematic identification of Plasmodium essential genes by transposon shuttle mutagenesis

After the deciphering of the genome sequences of several Plasmodium species, efforts must turn to elucidating gene function and identifying essential gene products. However, random approaches are lacking and gene targeting is inefficient in Plasmodium. Here, we established shuttle transposon mutagenesis in Plasmodium berghei. We constructed a mini-Tn5 derivative that can transpose into parasite genes cloned in Escherichia coli, providing an efficient means of generating knockout fragments. A 104-fold increase in frequencies of double-crossover homologous recombination in the parasite using a new electroporation technology permits to reproducibly generate pools of distinct mutants after transfection with mini-Tn5-interrupted sequences. The procedure opens the way to the systematic identification of essential genes in Plasmodium.

Differential Recognition of Citrate and a Metal-Citrate Complex by the Bacterial Chemoreceptor Tcp

The chemoreceptor Tcp of Salmonella enterica serovar Typhimurium can sense citrate and a metal-citrate complex as distinct attractants. In this study, we tried to investigate the molecular mechanism of this discrimination. That citrate binds directly to Tcp was verified by the site-specific thiol modification assays using membrane fractions prepared from Escherichia coli cells expressing the mutant Tcp receptors in which single Cys residues were introduced at positions in the putative ligand-binding pocket. To determine the region responsible for the ligand discrimination, we screened for mutations defective in taxis to magnesium in the presence of citrate. All of the isolated mutants from random mutagenesis with hydroxylamine were defective in both citrate and metal-citrate sensing, and the mutated residues are located in or near the α1-α2 and α3-α4 loops within the periplasmic domain. Further analyses with site-directed replacements around these regions demonstrated that the residue Asn67, which is presumed to lie at the subunit interface of the Tcp homodimer, plays a critical role in the recognition of the metal-citrate complex but not that of citrate. Various amino acids at this position differentially affect the citrate and metal-citrate sensing abilities. Thus, for the first time, the abilities to sense the two attractants were genetically dissected. Based on the results obtained in this study, we propose models in which the discrimination of the metal-citrate complex from citrate involves cooperative interaction at Asn67 and allosteric switching.

Context-Sensitive Grammar Transform: Compression and Pattern Matching

A framework of context-sensitive grammar transform is proposed. A greedy compression algorithm with the transform model is presented as well as a Knuth-Morris-Pratt (KMP)-type compressed pattern matching (CPM) algorithm. The compression performance is a match for gzip and Re-Pair. The search speed of our CPM algorithm is almost twice faster than the KMP type CPM algorithm on Byte-Pair-Encoding by Shibata et al. (2000), and in the case of short patterns, faster than the Boyer-Moore-Horspool algorithm with the stopper encoding by Rautio et al. (2002), which is regarded as one of the best combinations that allows a practically fast search.

The Reactions of Heme- and Verdoheme-Heme Oxygenase-1 Complexes with FMN-depleted NADPH-cytochrome P450 Reductase ELECTRONS REQUIRED FOR VERDOHEME OXIDATION CAN BE TRANSFERRED THROUGH A PATHWAY NOT INVOLVING FMN

Electrons utilized in the heme oxygenase (HO) reaction are provided by NADPH-cytochrome P450 reductase (CPR). To investigate the electron transfer pathway from CPR to HO, we examined the reactions of heme and verdoheme, the second intermediate in the heme degradation, complexed with rat HO-1 (rHO-1) using a rat FMN-depleted CPR; the FMN-depleted CPR was prepared by dialyzing the CPR mutant, Y140A/Y178A, against 2 m KBr. Degradation of heme in complex with rHO-1 did not occur with FMN-depleted CPR, notwithstanding that the FMN-depleted CPR was able to associate with the heme-rHO-1 complex with a binding affinity comparable with that of the wild-type CPR. Thus, the first electron to reduce the ferric iron of heme complexed with rHO-1 must be transferred from FMN. In contrast, verdoheme was converted to the ferric biliverdin-iron chelate with FMN-depleted CPR, and this conversion was inhibited by ferricyanide, indicating that electrons are certainly required for conversion of verdoheme to a ferric biliverdin-iron chelate and that they can be supplied from the FMN-depleted CPR through a pathway not involving FMN, probably via FAD. This conclusion was supported by the observation that verdoheme dimethyl esters were accumulated in the reaction of the ferriprotoporphyrin IX dimethyl ester-rHO-1 complex with the wild-type CPR. Ferric biliverdin-iron chelate, generated with the FMN-depleted CPR, was converted to biliverdin by the addition of the wild-type CPR or desferrioxamine. Thus, the final electron for reducing ferric biliverdin-iron chelate to release ferrous iron and biliverdin is apparently provided by the FMN of CPR.

Functional analyses of Pseudomonas putida benzoate transporters expressed in the yeast Saccharomyces cerevisiae.

Pseudomonas putida benF, benK, benE1, and benE2 genes encode proteins belonging to benzoate transporter super family, but those functions have not yet been elucidated. In this study we analyzed the functions of these gene products using the yeast Saccharomyces cerevisiae. P. putida gene products expressed in yeast cells were localized to the yeast plasma membrane and were involved in taking up benzoate into the cells. According to the sensitivity of yeast cell-growth to benzoate, it is proposed that benK, benE1, and benE2 gene products function as transporters, that take up benzoate into the cells, whereas the benF gene product functions as an efflux pump of benzoate.

Identification of Compounds with Potential Antibacterial Activity against Mycobacterium through Structure-Based Drug Screening

To identify novel antibiotics against Mycobacterium tuberculosis, we performed a hierarchical structure-based drug screening (SBDS) targeting the enoyl-acyl carrier protein reductase (InhA) with a compound library of 154,118 chemicals. We then evaluated whether the candidate hit compounds exhibited inhibitory effects on the growth of two model mycobacterial strains: Mycobacterium smegmatis and Mycobacterium vanbaalenii. Two compounds (KE3 and KE4) showed potent inhibitory effects against both model mycobacterial strains. In addition, we rescreened KE4 analogs, which were identified from a compound library of 461,383 chemicals through fingerprint analysis and genetic algorithm-based docking simulations. All of the KE4 analogs (KES1-KES5) exhibited inhibitory effects on the growth of M. smegmatis and/or M. vanbaalenii. Based on the predicted binding modes, we probed the structure-activity relationships of KE4 and its analogs and found a correlative relationship between the IC50 values and the interaction residues/LogP values. The most potent inhibitor, compound KES4, strongly and stably inhibited the long-term growth of the model bacteria and showed higher inhibitory effects (IC50 = 4.8 μM) than isoniazid (IC50 = 5.4 μM), which is a first-line drug for tuberculosis therapy. Moreover, compound KES4 did not exhibit any toxic effects that impede cell growth in several mammalian cell lines and enterobacteria. The structural and experimental information of these novel chemical compounds will likely be useful for the development of new anti-TB drugs. Furthermore, the methodology that was used for the identification of the effective chemical compound is also likely to be effective in the SBDS of other candidate medicinal drugs.

Development of a heme sensor using fluorescently labeled heme oxygenase-1

Free heme, the protein-unbound form of heme, has both toxic and regulatory effects on cells. To detect free heme at low concentrations, we developed a heme sensor using fluorescently labeled heme oxygenase-1 (HO-1), an enzyme that catalyzes oxidative heme degradation and has a high affinity for heme. The response of the heme sensor is based on the fluorescence quenching that occurs when heme binds to the enzyme. Each of the three fluorescently labeled HO-1s exhibits a 1:1 binding stoichiometry and an absorption spectrum similar to that of the heme complex of the wild-type HO-1. Titration of the labeled proteins with hemin resulted in fluorescence quenching in a hemin concentration-dependent manner, presumably due to an energy transfer from the fluorophore to the heme bound to HO-1. The sensor showed a potent affinity for heme with a dissociation constant in the low nanomolar range and a high selectivity for heme. Based on the linear response of the sensor to heme, we performed a fluorometric microplate assay. The sensor was able to selectively detect free heme but did not respond to heme bound to native hemoglobin. This assay will be a useful tool for determination of free heme in biological samples containing protein-bound heme.

Discovery of InhA inhibitors with anti-mycobacterial activity through a matched molecular pair approach.

The Mycobacterium tuberculosis (M. tuberculosis) enoyl-acyl carrier protein reductase (mtInhA) is an attractive enzyme and a thoroughly studied target for tuberculosis therapy. In this study, to identify novel structure-activity relationships (SARs) of mtInhA inhibitors, a series of diphenyl ether derivatives were designed based on the matched molecular pair (MMP) method, and the binding energies of these compounds were subsequently estimated by in silico structure-based drug screening (SBDS) to provide more useful data. Consequently, the 10 unique candidate compounds (KEM1-KEM10) were identified and assessed for the inhibition of mtInhA enzymatic activity, in vitro antibiotic effects against model mycobacteria and toxicity level on both intestinal bacteria and mammalian cells. Among the compounds tested, phenyl group (KEM4) and 2-fluorobenzyl group (KEM7) substitutions produced preferable inhibitory effects on mtInhA enzymatic activity relative to those provided by a furyl group (KES4: base compound) at the terminal of the compound, and KEM7 inhibited the growth of the mycobacteria strain with a lower IC50 value. Moreover, most of the candidate compounds exhibited neither inhibition of the growth of enterobacteria nor toxic effects on mammalian cells, though KEM10 exhibited toxicity against cultured MDCK cells. The structural and experimental information concerning these mtInhA inhibitors identified through MMP-based in silico screening will likely contribute to the lead optimisation of novel antibiotics for M. tuberculosis.