A cutinase-type polyesterase from Saccharomonospora viridis AHK190 (Cut190) has been shown to degrade the inner block of polyethylene terephthalate. A unique feature of Cut190 is that its function and stability are regulated by Ca(2+) binding. Our previous crystal structure analysis of Cut190S226P showed that one Ca(2+) binds to the enzyme, which induces large conformational changes in several loop regions to stabilize an open conformation [Miyakawa, T., et al. (2015) Appl. Microbiol. Biotechnol. 99, 4297]. In this study, to analyze the substrate recognition mechanism of Cut190, we determined the crystal structure of the inactive form of a Cut190 mutant, Cut190*S176A, in complex with calcium ions and/or substrates. We found that three calcium ions bind to Cut190*S176A, which is supported by analysis using native mass spectrometry experiments and 3D Reference Interaction Site Model calculations. The complex structures with the two substrates, monoethyl succinate and monoethyl adipate (engaged and open forms), presumably correspond to the pre- and post-reaction states, as the ester bond is close to the active site and pointing outward from the active site, respectively, for the two complexes. Ca(2+) binding induces the pocket to open, enabling the substrate to access the pocket more easily. Molecular dynamics simulations suggest that a post-reaction state in the engaged form presumably exists between the experimentally observed forms, indicating that the substrate would be cleaved in the engaged form and then requires the enzyme to change to the open form to release the product, a process that Ca(2+) can greatly accelerate.
INTRODUCTION: When treating ocular myasthenia gravis (MG), the risk/benefit profile of corticosteroids is unclear, and acetylcholinesterase inhibitors are not very effective. We examined the efficacy of topical naphazoline in the treatment of myasthenic blepharoptosis. METHODS: Sixty MG patients with blepharoptosis (32 with ocular symptoms only and 28 with mild generalized symptoms) were enrolled in a multicenter open trial of topical naphazoline. The effects were reported by patients via a questionnaire and were also confirmed for each patient at the clinic. RESULTS: Among 70 eyes of 60 patients, 20 eyes (28.6%) of 17 patients (28.3%) exhibited a marked response (full eye opening), and 24 eyes (34.3%) of 20 patients (33.3%) showed a good response (adequate but incomplete eye opening). Topical naphazoline was evaluated as useful in the treatment of myasthenic blepharoptosis by >70% of the patients. CONCLUSIONS: Topical naphazoline was found to be an effective supplementary symptomatic treatment for myasthenic blepharoptosis.
Bacillus subtilis is the best-characterized member of the Gram-positive bacteria. Its genome of 4,214,810 base pairs comprises 4,100 protein-coding genes. Of these protein-coding genes, 53% are represented once, while a quarter of the genome corresponds to several gene families that have been greatly expanded by gene duplication, the largest family containing 77 putative ATP-binding transport proteins. In addition, a large proportion of the genetic capacity is devoted to the utilization of a variety of carbon sources, including many plant-derived molecules. The identification of five signal peptidase genes, as well as several genes for components of the secretion apparatus, is important given the capacity of Bacillus strains to secrete large amounts of industrially important enzymes. Many of the genes are involved in the synthesis of secondary metabolites, including antibiotics, that are more typically associated with Streptomyces species. The genome contains at least ten prophages or remnants of prophages, indicating that bacteriophage infection has played an important evolutionary role in horizontal gene transfer, in particular in the propagation of bacterial pathogenesis.
        
Title: The Bacillus subtilis chromosome region near 78 degrees contains the genes encoding a new two-component system, three ABC transporters and a lipase Yamamoto H, Uchiyama S, Sekiguchi J Ref: Gene, 181:147, 1996 : PubMed
The nucleotide sequence of a 9444-bp segment around the 78 degrees region of the Bacillus subtilis (Bs) chromosome has been determined. Nine putative orfs were identified. The deduced amino acid sequences of the products of two of them (yfiJ and yfiK) exhibit high similarity to those of a sensor protein, DegS, and a transcriptional regulatory protein, DegU, of Bs, respectively. Three of them (yfiL, yfiM and yfiN) seem to be ABC transporter genes. One orf (designated as lipB), the closest to the sspE among the nine orfs, is the second lipase gene in Bs.
        
Title: Cloning and sequencing of a 27.8-kb nucleotide sequence of the 79 degrees-81 degrees region of the Bacillus subtilis genome containing the sspE locus Yamamoto H, Uchiyama S, Sekiguchi J Ref: DNA Research, 3:257, 1996 : PubMed
The nucleotide sequence of a 27830-bp DNA segment in the 79 degrees-81 degrees region of the Bacillus subtilis genome has been determined. This region contains 29 complete ORFs including the sspE gene, which encodes a small acid-soluble spore protein gamma and locates on the one side terminal of our assigned region. A homology search for the products deduced from the 29 ORFs revealed that nine of them exhibit significant similarity to known proteins, e.g. proteins involved in an iron uptake system, a multidrug resistance protein, a chloramphenicol resistance protein, epoxide hydrolase, adenine glycosylase, and a glucose-1-dehydrogenase homolog.