Mining prokaryotic genomes for unknown amino acids: a stop-codon-based approach

<p>Abstract</p> <p>Background</p> <p>Selenocysteine and pyrrolysine are the 21st and 22nd amino acids, which are genetically encoded by stop codons. Since a number of microbial genomes have been completely sequenced to date, it is tempting to ask whether the 23rd amino acid is left undiscovered in these genomes. Recently, a computational study addressed this question and reported that no tRNA gene for unknown amino acid was found in genome sequences available. However, performance of the tRNA prediction program on an unknown tRNA family, which may have atypical sequence and structure, is unclear, thereby rendering their result inconclusive. A protein-level study will provide independent insight into the novel amino acid.</p> <p>Results</p> <p>Assuming that the 23rd amino acid is also encoded by a stop codon, we systematically predicted proteins that contain stop-codon-encoded amino acids from 191 prokaryotic genomes. Since our prediction method relies only on the conservation patterns of primary sequences, it also provides an opportunity to search novel selenoproteins and other readthrough proteins. It successfully recovered many of currently known selenoproteins and pyrrolysine proteins. However, no promising candidate for the 23rd amino acid was detected, and only one novel selenoprotein was predicted.</p> <p>Conclusion</p> <p>Our result suggests that the unknown amino acid encoded by stop codons does not exist, or its phylogenetic distribution is rather limited, which is in agreement with the previous study on tRNA. The method described here can be used in future studies to explore novel readthrough events from complete genomes, which are rapidly growing.</p

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

システインデスルフラーゼとセレノシステインリアーゼの酵素学的研究

京都大学0048新制・課程博士博士(農学)甲第8031号農博第1081号新制||農||789(附属図書館)学位論文||H11||N3326(農学部図書室)UT51-99-T742京都大学大学院農学研究科農芸化学専攻(主査)教授 江﨑 信芳, 教授 清水 昌, 教授 關谷 次郎学位規則第4条第1項該当Doctor of Agricultural ScienceKyoto UniversityDFA

Cysteine Sulfinate Desulfinase, a NIFS-like Protein of Escherichia coli with Selenocysteine Lyase and Cysteine Desulfurase Activities: Gene Cloning, Purification and Characterization of a Novel Pyridoxal Enzyme

Selenocysteine lyase (EC 4.4.1.16) exclusively decomposes selenocysteine to alanine and elemental selenium whereas cysteine desulfurase (NIFS protein) of Azotobacter vinelandii acts indiscriminately on both cysteine and selenocysteine to produce elemental sulfur and selenium respectively, and alanine. These proteins exhibit some sequence homology. The Escherichia coli genome contains three genes with sequence homology to nifS. We have cloned the gene mapped at 63.4 min in the chromosome, and have expressed, purified to homogeneity, and characterized the gene product. The enzyme comprises two identical subunits with 401 amino acid residues (Mr 43,238) and contains pyridoxal 5'-phosphate as a coenzyme. The enzyme catalyzes the removal of elemental sulfur and selenium atoms from L-cysteine, L-cystine, L-selenocysteine and L-selenocystine to produce L-alanine. Because L-cysteine sulfinic acid was desulfinated to form L-alanine as the preferred substrate, we have named this new enzyme cysteine sulfinate desulfinase. Mutant enzymes having alanine substituted for each of the four cysteinyl residues were all active. Cys358 corresponds to Cys325 of A. vinelandii NIFS, which is conserved among all NIFS-like proteins and catalytically essential is not required for cysteine sulfinate desulfinase. Thus, the enzyme is distinct from A. vinelandii NIFS in this respect

Characterization of a thermostable 2,4-diaminopentanoate dehydrogenase from Fervidobacterium nodosum Rt17-B1.

2, 4-Diaminopentanoate dehydrogenase (2, 4-DAPDH), which is involved in the oxidative ornithine degradation pathway, catalyzes the NAD(+)- or NADP(+)-dependent oxidative deamination of (2R, 4S)-2, 4-diaminopentanoate (2, 4-DAP) to form 2-amino-4-oxopentanoate. A Fervidobacterium nodosum Rt17-B1 gene, Fnod_1646, which codes for a protein with sequence similarity to 2, 4-DAPDH discovered in metagenomic DNA, was cloned and overexpressed in Escherichia coli, and the gene product was purified and characterized. The purified protein catalyzed the reduction of NAD(+) and NADP(+) in the presence of 2, 4-DAP, indicating that the protein is a 2, 4-DAPDH. The optimal pH and temperature were 9.5 and 85°C, respectively, and the half-denaturation time at 90°C was 38 min. Therefore, the 2, 4-DAPDH from F. nodosum Rt17-B1 is an NAD(P)(+)-dependent thermophilic-alkaline amino acid dehydrogenase. This is the first thermophilic 2, 4-DAPDH reported, and it is expected to be useful for structural and functional analyses of 2, 4-DAPDH and for the enzymatic production of chiral amine compounds. Activity of 2, 4-DAPDH from F. nodosum Rt17-B1 was suppressed by 2, 4-DAP via uncompetitive substrate inhibition. In contrast, the enzyme showed typical Michaelis-Menten kinetics toward 2, 5-diaminohexanoate. The enzyme was uncompetitively inhibited by d-ornithine with an apparent Ki value of 0.1 mM. These results suggest a regulatory role for this enzyme in the oxidative ornithine degradation pathway

Characterization of a Novel Porin-Like Protein, ExtI, from Geobacter sulfurreducens and Its Implication in the Reduction of Selenite and Tellurite

The extI gene in Geobacter sulfurreducens encodes a putative outer membrane channel porin, which resides within a cluster of extHIJKLMNOPQS genes. This cluster is highly conserved across the Geobacteraceae and includes multiple putative c-type cytochromes. In silico analyses of the ExtI sequence, together with Western blot analysis and proteinase protection assays, showed that it is an outer membrane protein. The expression level of ExtI did not respond to changes in osmolality and phosphate starvation. An extI-deficient mutant did not show any significant impact on fumarate or Fe(III) citrate reduction or sensitivity to β-lactam antibiotics, as compared with those of the wild-type strain. However, extI deficiency resulted in a decreased ability to reduce selenite and tellurite. Heme staining analysis revealed that extI deficiency affects certain heme-containing proteins in the outer and inner membranes, which may cause a decrease in the ability to reduce selenite and tellurite. Based on these observations, we discuss possible roles for ExtI in selenite and tellurite reduction in G. sulfurreducens

Escherichia coli Dihydropyrimidine Dehydrogenase Is a Novel NAD-Dependent Heterotetramer Essential for the Production of 5,6-Dihydrouracil▿

The reductive pyrimidine catabolic pathway is absent in Escherichia coli. However, the bacterium contains an enzyme homologous to mammalian dihydropyrimidine dehydrogenase. Here, we show that E. coli dihydropyrimidine dehydrogenase is the first member of a novel NADH-dependent subclass of iron-sulfur flavoenzymes catalyzing the conversion of uracil to 5,6-dihydrouracil in vivo