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185 results on '"Uno, Tomohide"'

19. Reciprocal size-effect relationship of the key residues in determining regio- and stereospecificities of DHEA hydroxylase activity in P450 2a5

Author

Uno, Tomohide, Mitchell, Eddy, Aida, Kaoru, Lambert, Mill H., Darden, Thomas A., Pedersen, Lee G., and Negishi, Masahiko

Subjects
Enzymes -- Structure-activity relationship, Dehydroepiandrosterone -- Analysis, Hydroxylases -- Analysis, Biological sciences, Chemistry
Abstract

The role of the side-chain size of residues 117, 209 and 481 in murine P450 (mP450) 2a4/2a5 system hydroxylates DHEA was analyzed by bacterial expression and site-directed mutagenesis of mP450 2a5 from wild Mus minutoides. Analysis of the effects of hydrophobic amino acid side-chain size on P450 activity indicated the presence of a reciprocal size-effect relationship that affects the specific activities of P role of the residue's size in defining P450 activity. Furthermore, the size of amino acid side chains altered the regio- and stereospecificities of steroid hydroxylase activity in the P450s.

Published
1997

26. Molecular and functional characterization of the American cockroach, Periplaneta americana, Rab5: the first exopterygotan low molecular weight ovarian GTPase during oogenesis.

Author

Elmogy, Mohamed, Mohamed, Amr A., Tufail, Muhammad, Uno, Tomohide, and Takeda, Makio

Subjects
AMERICAN cockroach, MESSENGER RNA, MOLECULAR weights, OOGENESIS, IMMUNOFLUORESCENCE
Abstract

Abstract: The small Rab GTPases are key regulators of membrane vesicle trafficking. Ovaries of Periplaneta americana (Linnaeus) (Blattodea: Blattidae) have small molecular weight GTP/ATP‐binding proteins during early and late vitellogenic periods of oogenesis. However, the identification and characterization of the detected proteins have not been yet reported. Herein, we cloned a cDNA encoding Rab5 from the American cockroach, P. americana, ovaries (PamRab5). It comprises 796 bp, encoding a protein of 213 amino acid residues with a predicted molecular weight of 23.5 kDa. PamRab5 exists as a single‐copy gene in the P. americana genome, as revealed by Southern blot analysis. An approximate 2.6 kb ovarian mRNA was transcribed especially at high levels in the previtellogenic ovaries, detected by Northern blot analysis. The muscle and head tissues also showed high levels of PamRab5 transcript. PamRab5 protein was localized, via immunofluorescence labeling, to germline‐derived cells of the oocytes, very early during oocyte differentiation. Immunoblotting detected a ∼25 kDa signal as a membrane‐associated form revealed after application of detergent in the extraction buffer, and 23 kDa as a cytosolic form consistent with the predicted molecular weight from amino acid sequence in different tissues including ovary, muscles and head. The PamRab5 during late vitellogenic periods is required to regulate the endocytotic machinery during oogenesis in this cockroach. This is the first report on Rab5 from a hemimetabolan, and presents an inaugural step in probing the molecular premises of insect oocyte endocytotic trafficking important for oogenesis and embryonic development. [ABSTRACT FROM AUTHOR]

Published
2018
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29. Functional characterization of CYP52G3 from Aspergillus oryzae and its application for bioconversion and synthesis of hydroxyl flavanone and steroids.

Author

Uno, Tomohide, Yanase, Takeshi, and Imaishi, Hiromasa

Subjects
*KOJI, *STEROIDS, *HYDROXYL group, *FLAVANONES, *CYTOCHROME P-450
Abstract

Aspergillus oryzae is a fungus widely used in traditional Japanese fermentation industries. Cytochrome P450 (CYP) proteins are ubiquitously distributed in nature and display a broad range of enzymatic activities. A novel CYP52 (CYP52G3) gene was found in A. oryzae. In this study, we report the functional characterization of CYP52G3. The recombinant protein was expressed heterologously in Escherichia coli, and its membrane fraction isolated. CYP52G3 showed activities for 7-ethoxycoumarin and α-naphtoflavone. Furthermore, CYP52G3 hydroxylated flavanone at the 4′ and 6 position and metabolized some hydroxyl-flavanones and steroids. Bioconversion experiments indicated that CYP52G3 could convert flavanone and testosterone in a synthetic medium. The conversion rates of flavanone and testosterone at 24 H were 50% and 70%, respectively. These results support that CYP52G3 could prove a useful enzyme for the efficient production of new compounds from flavonoids and steroids. [ABSTRACT FROM AUTHOR]

Published
2017
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