Understanding the Molecular Mechanism Underlying the High Catalytic Activity of p-Hydroxybenzoate Hydroxylase Mutants for Producing Gallic Acid.

p-Hydroxybenzoate hydroxylase (PHBH) is a flavoprotein monooxygenase that catalyzes the hydroxylation of p-hydroxybenzoate (p-OHB) to 3,4-dihydroxybenzoate (3,4-DOHB). PHBH can bind to other benzoate derivatives in addition to p-OHB; however, hydroxylation does not occur on 3,4-DOHB. Replacement of Tyr385 with Phe forms a mutant, which enables the production of 3,4,5-trihydroxybenzonate (gallic acid) from 3,4-DOHB, although the catalytic activity of the mutant is quite low. In this study, we report how the L199V/Y385F double mutant exhibits activity for producing gallic acid 4.3-fold higher than that of the Y385F single mutant. This improvement in catalytic activity is primarily due to the suppression of a shunt reaction that wastes reduced nicotinamide adenine dinucleotide phosphate by producing H2O2. To further elucidate the molecular mechanism underlying this higher catalytic activity, we performed molecular dynamics simulations and quantum mechanics/molecular mechanics calculations, in addition to determining the crystal structure of the Y385F·3,4-DOHB complex. The simulations showed that the Y385F mutation facilitates the deprotonation of the 4-hydroxy group of 3,4-DOHB, which is necessary for initiating hydroxylation. Moreover, the L199V mutation in addition to the Y385F mutation allows the OH moiety in the peroxide group of C-(4a)-flavin hydroperoxide to come into the proximity of the C5 atom of 3,4-DOHB. Overall, this study provides a consistent explanation for the change in the catalytic activity of PHBH caused by mutations, which will enable us to better design an enzyme with different activities.

Complete sequencing and characterization of 21,243 full-length human cDNAs.

As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs.

A reporter assay for G-protein-coupled receptors using a B-cell line suitable for stable episomal expression.

We have established a cAMP response element (CRE)-mediated reporter assay system for G-protein-coupled receptors (GPCRs) using an oriP-based estrogen-inducible expression vector and the B-cell line (GBC53 or GBCC71) that expresses EBNA-1 and is adapted to serum-free culture. GBC53 harbors a GAL4-ER expression unit and a CRE-luciferase gene in the genome, and GBCC71 also harbors expression units for two chimeric Galphas proteins (Gs/q and Gs/i). Introduction of a GPCR expression plasmid into GBC53 or GBCC71 creates polyclonal stable transformants in 2 weeks, and these are easily expanded and used for assays after induction of the GPCR expression. Using GBC53, we detected ligand-dependent signals of Gs-coupled GPCRs such as glucagon-like peptide 1 receptor (GLP1R) and beta2 adrenergic receptor (beta2AR) with high sensitivity. Interestingly, we also detected constitutive activity of beta2AR. Using GBCC71, we detected ligand-dependent signals of Gq- or Gi-coupled GPCRs such as H1 histamine receptor and CXCR1 chemokine receptor in addition to Gs-coupled GPCRs. An agonist, antagonist, or inverse agonist was successfully evaluated in this system. We succeeded in constructing a 384-well high-throughput screening (HTS) system for GLP1R. This system enabled us to easily and rapidly make a large number of efficient GPCR assay systems suitable for HTS as well as ligand hunting of orphan GPCRs.

Characterization of orphan monooxygenases by rapid substrate screening using FT-ICR mass spectrometry.

Characterization of orphan enzymes, for which the catalytic functions and actual substrates are still not elucidated, is a significant challenge in the postgenomic era. Here, we describe a general strategy for exploring the catalytic potentials of orphan monooxygenases based on direct infusion analysis by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR/MS). Eight cytochromes P450 from Bacillus subtilis were recombinantly expressed in Escherichia coli and subjected to a reconstitution system containing appropriate electron transfer components and many potential substrates. The reaction mixtures were directly analyzed using FT-ICR/MS, and substrates of the putative enzymes were readily identified from the mass spectral data. This allowed identification of previously unreported CYP109B1 substrates and the functional assignment of two putative cytochromes P450, CYP107J1 and CYP134A1. The FT-ICR/MS-based approach can be easily applied to large-scale screening with the aid of the extremely high mass resolution and accuracy.

2,3-Dihydroxybenzoate meta-Cleavage Pathway is Involved in o-Phthalate Utilization in Pseudomonas sp. strain PTH10.

Pseudomonas sp. strain PTH10 can utilize o-phthalate which is a key intermediate in the bacterial degradation of some polycyclic aromatic hydrocarbons. In this strain, o-phthalate is degraded to 2,3-dihydroxybenzoate and further metabolized via the 2,3-dihydroxybenzoate meta-cleavage pathway. Here, the opa genes which are involved in the o-phthalate catabolism were identified. Based on the enzymatic activity of the opa gene products, opaAaAbAcAd, opaB, opaC, and opaD were found to code for o-phthalate 2,3-dioxygenase, dihydrodiol dehydrogenase, 2,3-dihydroxybenzoate 3,4-dioxygenase, and 3-carboxy-2-hydroxymuconate-6-semialdehyde decarboxylase, respectively. Collectively, these enzymes are thought to catalyze the conversion of o-phthalate to 2-hydroxymuconate-6-semialdehyde. Deletion mutants of the above opa genes indicated that their products were required for the utilization of o-phthalate. Transcriptional analysis showed that the opa genes were organized in the same transcriptional unit. Quantitative analysis of opaAa, opaB, opaC, opaD, opaE, and opaN revealed that, except for opaB and opaC, all other genes were transcriptionally induced during growth on o-phthalate. The constitutive expression of opaB and opaC, and the transcriptional induction of opaD located downstream of opaB, suggest several possible internal promoters are existence in the opa cluster. Together, these results strongly suggest that the opa genes are involved in a novel o-phthalate catabolic pathway in strain PTH10.

GeneLook: a novel ab initio gene identification system suitable for automated annotation of prokaryotic sequences.

With the rapid increases in the amounts of sequence data for prokaryotic genomes, it has become important to develop systems for automated and accurate genome annotation. We present herein a novel ab initio gene identification system, GeneLook, that predicts protein-coding open reading frames (ORFs) with high sensitivity and specificity with no prior knowledge of the sequence composition. The system predicts protein-coding ORFs in two stages, seed ORF selection and main prediction. In the selection of reliable seed ORFs containing at least 200 codons, GeneLook predicts translation start sites and operon structures through searches for ribosome-binding sites and a novel operon prediction algorithm. The codon and nucleotide frequencies of seed ORFs are then used to determine values for two new coding-potential parameters for identification of protein-coding ORFs of at least 34 codons and for another parameter that improves the prediction accuracy for GC-rich genomes. In the main prediction, GeneLook uses these parameters to identify the most likely genes of a given minimal length. We assessed the performance of GeneLook with two indices, sensitivity and specificity that are defined as true positives (TP)/(TP+false negatives) and TP/(TP+false positives), respectively. This system predicted protein-coding ORFs for Escherichia coli and Bacillus subtilis with sensitivities of 96.5% and 96.2%, respectively, and specificities of 96.9% and 96.1%, respectively. The system also identified 94.1% of annotated genes of the Pseudomonas aeruginosa genome, which is GC-rich, with high specificity (97.2%). Furthermore, GeneLook identified protein-coding ORFs with high accuracy from a wide variety of prokaryotic genomes.

HPLC Retention time prediction for metabolome analysi.

Liquid Chromatography Time-of-Flight Mass Spectrometry (LC-TOF-MS) is widely used for profiling metabolite compounds. LC-TOF-MS is a chemical analysis technique that combines the physical separation capabilities of high-pressure liquid chromatography (HPLC) with the mass analysis capabilities of Time-of-Flight Mass Spectrometry (TOF-MS) which utilizes the difference in the flight time of ions due to difference in the mass-to-charge ratio. Since metabolite compounds have various chemical characteristics, their precise identification is a crucial problem of metabolomics research. Contemporaneously analyzed reference standards are commonly required for mass spectral matching and retention time matching, but there are far fewer reference standards than there are compounds in the organism. We therefore developed a retention time prediction method for HPLC to improve the accuracy of identification of metabolite compounds. This method uses a combination of Support Vector Regression and Multiple Linear Regression adaptively to the measured retention time. We achieved a strong correlation (correlation coefficient = 0.974) between measured and predicted retention times for our experimental data. We also demonstrated a successful identification of an E. coli metabolite compound that cannot be identified by precise mass alone.

Complete genome sequence and comparative analysis of Shewanella violacea, a psychrophilic and piezophilic bacterium from deep sea floor sediments.

Remineralization of organic matter in deep-sea sediments is important in oceanic biogeochemical cycles, and bacteria play a major role in this process. Shewanella violacea DSS12 is a psychrophilic and piezophilic gamma-proteobacterium that was isolated from the surface layer of deep sea sediment at a depth of 5110 m. Here, we report the complete genome sequence of S. violacea and comparative analysis with the genome of S. oneidensis MR-1, isolated from sediments of a freshwater lake. Unlike S. oneidensis, this deep-sea Shewanella possesses very few terminal reductases for anaerobic respiration and no c-type cytochromes or outer membrane proteins involved in respiratory Fe(iii) reduction, which is characteristic of most Shewanella species. Instead, the S. violacea genome contains more terminal oxidases for aerobic respiration and a much greater number of putative secreted proteases and polysaccharases, in particular, for hydrolysis of collagen, cellulose and chitin, than are encoded in S. oneidensis. Transporters and assimilatory reductases for nitrate and nitrite, and nitric oxide-detoxifying mechanisms (flavohemoglobin and flavorubredoxin) are found in S. violacea. Comparative analysis of the S. violacea genome revealed the respiratory adaptation of this bacterium to aerobiosis, leading to predominantly aerobic oxidation of organic matter in surface sediments, as well as its ability to efficiently use diverse organic matter and to assimilate inorganic nitrogen as a survival strategy in the nutrient-poor deep-sea floor.