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We performed mitogenome analysis of Japanese firefly Luciola cruciata (Coleoptera: Lampyridae), which is unique species in Japan. It is classified into six haplotypes based on the difference of COII sequence on mitochondrial DNA. The complete mitogenome sequence of Tohoku group has been registered so far, we newly analysed West Japan groups which belong to different haplotype. The total length of analysed mitogenome was 15,990 bp, being one base longer than the case of Tohoku’s firefly. The base substitution was found at 273 positions over the whole mitochondrial sequence, while amino acid substitution accompanying it was observed at only 11 positions.

We will report the complete mitochondrial genome sequence of Japanese firefly ‘Heike Botaru’, Luciola lateralis (Coleoptera: Lampyridae). Total length of this mitogenome was 16,719 bp and the composition of each base was A (42.50%), C (9.01%), G (14.16%), T (34.33%), respectively. The obtained sequence fulfils general mitogenome composition of metazoan (13 protein coding sequences (CDSs), 22 tRNA genes, two rRNA subunits, and an AT-rich region). From the phylogenetic tree analysis using 25 kinds of insect mitogenome including firefly family was found that L. lateralis is the closest to the genus Aquatica.

Cucumisin [EC 3.4.21.25], a subtilisin-like serine endopeptidase, was isolated from melon fruit, Cucumis melo L. Mature cucumisin (67 kDa, 621 residues) is produced by removal of the propeptide (10 kDa, 88 residues) from the cucumisin precursor by subsequence processing. It is reported that cucumisin is inhibited by its own propeptide. The crystal structure of mature cucumisin is reported to be composed of three domains: the subtilisin-like catalytic domain, the protease-associated domain and the C-terminal fibronectin-III-like domain. In this study, the crystal structure of the mature cucumisin•propeptide complex was determined by the molecular replacement method and refined at 1.95 Å resolution. In this complex, the propeptide had a domain of the α-β sandwich motif with four-stranded antiparallel β-sheets, two helices and a strand of the C-terminal region. The β-sheets of the propeptide bind to two parallel surface helices of cucumisin through hydrophobic interaction and 27 hydrogen bonds. The C-terminus of the propeptide binds to the cleft of the active site as peptide substrates. The inhibitory assay suggested that the C-terminal seven residues of the propeptide do not inhibit the cucumisin activity. The crystal structure of the cucumisin•propeptide complex revealed the regulation mechanism of cucumisin activity. [ABSTRACT FROM AUTHOR]

Acinetobacter sp. strain YAA has five genes (atdA1 to atdA5) involved in aniline oxidation as a part of the aniline degradation gene cluster. From sequence analysis, the five genes were expected to encode a glutamine synthetase (GS)-like protein (AtdA1), glutamine amidotransferase-like protein (AtdA2), and an aromatic compound dioxygenase (AtdA3, AtdA4, and AtdAS) (M. Takeo, T. Fujii, and Y. Maeda, 1. Ferment. Bioeng. 85:17-24, 1998). A recombinant Pseudomonas strain harboring these five g quantitatively converted aniline into catechol, demonstrating that catechol is the major oxidation product from aniline. To elucidate the function of the GS-like protein AtdA1 in aniline oxidation, we purified it from recombinant Escherichia coli harbor atdA1. The purified AtdA1 protein produced gamma-glutamylanilide (γ-GA) quantitatively from aniline and L-glutamate in the presence of ATP and MgC12. This reaction was identical to glutamine synthesis by GS, except for the use of aniline instead of ammonia as the substrate. Recombinant Pseudomonas strains harboring the dioxygenase genes (atdA3 to atdA5) were unable to degrade aniline but converted γ-GA into catechol, indicating that γ-GA is an intermediate to catechol and a direct substrate for the dioxygenase. Unexpectedly, a recombinant Pseudomonas strain harboring only atA2 hydrolyzed γ-GA into aniline, reversing the γ-GA formation by AtdA1. Deletion ofatdA2 from atdA1 to atdA5 caused γ-GA accumulation from aniline in recombinant Pseudomonas cells and inhibited the growth of a recombinant Acinetobacter strain on aniline, suggesting that AtdA2 prevents γ-GA accumulation that is harmful to the host cell. [ABSTRACT FROM AUTHOR]

We performed x-ray crystallographic analyses of the 6-aminohexanoate cyclic dimer (Acd) hydrolase (NyIA) from Arthrobacter sp., an enzyme responsible for the degradation of the nylon-6 industry byproduct. The fold adopted by the 472amino acid polypeptide generated a compact mixed α/β fold, typically found in the amidase signature superfamily; this fold was especially similar to the fold of glutamyl-tRNAGln amidotransferase subunit A (z score, 49.4) and malonamidase E2 (z score, 44.8). Irrespective of the high degree of structural similarity to the typical amidase signature superfamily enzymes, the specific activity of NyIA for glutamine, malonamide, and indoleacetamide was found to be lower than 0.5% of that for Acd. However, NyIA possessed carboxylesterase activity nearly equivalent to the Acd hydrolytic activity. Structural analysis of the inactive complex between the activity-deficient S174A mutant of NyIA and Acd, performed at 1.8 A resolution, suggested the following enzyme/substrate interactions: a Ser174-cis-Ser150-Lys72 triad constitutes the catalytic center; the backbone N in Ala'7' and Ala172 are involved in oxyanion stabilization; Cys316-Sγ forms a hydrogen bond with nitrogen (Acd-N7) at the uncleaved amide bond in two equivalent amide bonds of Acd. A single S174A, S15OA, or K72A substitution in NyIA by site-directed mutagenesis decreased the Acd hydrolytic and esterolytic activities to undetectable levels, indicating that Ser174-cis-Ser150-Lys72 is essential for catalysis. In contrast, substitutions at position 316 specifically affected Acd hydrolytic activity, suggesting that Cys316 is responsible for Acd binding. On the basis of the structure and functional analysis, we discussed the catalytic mechanisms and evolution of NyIA in comparison with other Ser-reactive hydrolases. [ABSTRACT FROM AUTHOR]