Mizohata E

References (3)

Title : Loop of Streptomyces Feruloyl Esterase Plays an Important Role in the Enzyme's Catalyzing the Release of Ferulic Acid from Biomass - Uraji_2018_Appl.Environ.Microbiol_84_
Author(s) : Uraji M , Tamura H , Mizohata E , Arima J , Wan K , Ogawa K , Inoue T , Hatanaka T
Ref : Applied Environmental Microbiology , 84 : , 2018
Abstract : Feruloyl esterases (FAEs) are key enzymes required for the production of ferulic acid from agricultural biomass. Previously, we identified and characterized R18, an FAE from Streptomyces cinnamoneus NBRC 12852, which showed no sequence similarity to the known FAEs. To determine the region involved in its catalytic activity, we constructed chimeric enzymes using R18 and its homolog (TH2-18) from S. cinnamoneus strain TH-2. Although R18 and TH2-18 showed 74% identity in their primary sequences, the recombinant proteins of these two FAEs (recombinant R18 [rR18] and rTH2-18) showed very different specific activities toward ethyl ferulate. By comparing the catalytic activities of the chimeras, a domain comprised of residues 140 to 154 was found to be crucial for the catalytic activity of R18. Furthermore, we analyzed the crystal structure of rR18 at a resolution of 1.5 A to elucidate the relationship between its activity and its structure. rR18 possessed a typical catalytic triad, consisting of Ser-191, Asp-214, and His-268, which was characteristic of the serine esterase family. By structural analysis, the above-described domain was found to be present in a loop-like structure (the R18 loop), which possessed a disulfide bond conserved in the genus Streptomyces Moreover, compared to rTH2-18 of its parental strain, the TH2-18 mutant, in which Pro and Gly residues were inserted into the domain responsible for forming the R18 loop, showed markedly high kcat values using artificial substrates. We also showed that the FAE activity of TH2-18 toward corn bran, a natural substrate, was improved by the insertion of the Gly and Pro residues.IMPORTANCEStreptomyces species are widely distributed bacteria that are predominantly present in soil and function as decomposers in natural environments. They produce various enzymes, such as carbohydrate hydrolases, esterases, and peptidases, which decompose agricultural biomass. In this study, based on the genetic information on two Streptomyces cinnamoneus strains, we identified novel feruloyl esterases (FAEs) capable of producing ferulic acid from biomass. These two FAEs shared high similarity in their amino acid sequences but did not resemblance any known FAEs. By comparing chimeric proteins and performing crystal structure analysis, we confirmed that a flexible loop was important for the catalytic activity of Streptomyces FAEs. Furthermore, we determined that the catalytic activity of one FAE was improved drastically by inserting only 2 amino acids into its loop-forming domain. Thus, differences in the amino acid sequence of the loop resulted in different catalytic activities. In conclusion, our findings provide a foundation for the development of novel enzymes for industrial use.
ESTHER : Uraji_2018_Appl.Environ.Microbiol_84_
PubMedSearch : Uraji_2018_Appl.Environ.Microbiol_84_
PubMedID: 29150515
Gene_locus related to this paper: strcj-estA

Title : Structural Basis for the Serratia marcescens Lipase Secretion System: Crystal Structures of the Membrane Fusion Protein and Nucleotide-Binding Domain - Murata_2017_Biochemistry_56_6281
Author(s) : Murata D , Okano H , Angkawidjaja C , Akutsu M , Tanaka SI , Kitahara K , Yoshizawa T , Matsumura H , Kado Y , Mizohata E , Inoue T , Sano S , Koga Y , Kanaya S , Takano K
Ref : Biochemistry , 56 :6281 , 2017
Abstract : Serratia marcescens secretes a lipase, LipA, through a type I secretion system (T1SS). The T1SS for LipA, the Lip system, is composed of an inner membrane ABC transporter with its nucleotide-binding domains (NBD), LipB, a membrane fusion protein, LipC, and an outer membrane channel protein, LipD. Passenger protein secreted by this system has been functionally and structurally characterized well, but relatively little information about the transporter complex is available. Here, we report the crystallographic studies of LipC without the membrane anchor region, LipC-, and the NBD of LipB (LipB-NBD). LipC- crystallographic analysis has led to the determination of the structure of the long alpha-helical and lipoyl domains, but not the area where it interacts with LipB, suggesting that the region is flexible without LipB. The long alpha-helical domain has three alpha-helices, which interacts with LipD in the periplasm. LipB-NBD has the common overall architecture and ATP hydrolysis activity of ABC transporter NBDs. Using the predicted models of full-length LipB and LipD, the overall structural insight into the Lip system is discussed.
ESTHER : Murata_2017_Biochemistry_56_6281
PubMedSearch : Murata_2017_Biochemistry_56_6281
PubMedID: 29094929

Title : Serial Femtosecond Crystallography and Ultrafast Absorption Spectroscopy of the Photoswitchable Fluorescent Protein IrisFP - Colletier_2016_J.Phys.Chem.Lett_7_882
Author(s) : Colletier JP , Sliwa M , Gallat FX , Sugahara M , Guillon V , Schiro G , Coquelle N , Woodhouse J , Roux L , Gotthard G , Royant A , Uriarte LM , Ruckebusch C , Joti Y , Byrdin M , Mizohata E , Nango E , Tanaka T , Tono K , Yabashi M , Adam V , Cammarata M , Schlichting I , Bourgeois D , Weik M
Ref : J Phys Chem Lett , 7 :882 , 2016
Abstract : Reversibly photoswitchable fluorescent proteins find growing applications in cell biology, yet mechanistic details, in particular on the ultrafast photochemical time scale, remain unknown. We employed time-resolved pump-probe absorption spectroscopy on the reversibly photoswitchable fluorescent protein IrisFP in solution to study photoswitching from the nonfluorescent (off) to the fluorescent (on) state. Evidence is provided for the existence of several intermediate states on the pico- and microsecond time scales that are attributed to chromophore isomerization and proton transfer, respectively. Kinetic modeling favors a sequential mechanism with the existence of two excited state intermediates with lifetimes of 2 and 15 ps, the second of which controls the photoswitching quantum yield. In order to support that IrisFP is suited for time-resolved experiments aiming at a structural characterization of these ps intermediates, we used serial femtosecond crystallography at an X-ray free electron laser and solved the structure of IrisFP in its on state. Sample consumption was minimized by embedding crystals in mineral grease, in which they remain photoswitchable. Our spectroscopic and structural results pave the way for time-resolved serial femtosecond crystallography aiming at characterizing the structure of ultrafast intermediates in reversibly photoswitchable fluorescent proteins.
ESTHER : Colletier_2016_J.Phys.Chem.Lett_7_882
PubMedSearch : Colletier_2016_J.Phys.Chem.Lett_7_882
PubMedID: 26866390