Distinct enzymatic and cellular characteristics of two secretory phospholipases A2 in the filamentous fungus Aspergillus oryzae |
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| Authors: | Tomoyuki Nakahama Yoshito Nakanishi Arturo R Viscomi Kohei Takaya Katsuhiko Kitamoto Simone Ottonello Manabu Arioka |
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| Institution: | 1. Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan;2. Department of Biochemistry and Molecular Biology, University of Parma, Viale G. P. Usberti 23/A, I-43100 Parma, Italy;1. Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, GA 30602, United States;2. Department of Drug Discovery & Development, Harrison School of Pharmacy, Auburn University, Auburn, AL 36849-5503, United States;1. Bio-energy Corporation, Research and Development Laboratory, 2-9-7 Minaminanamatsu, Amagasaki 660-0053, Japan;2. Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan;1. Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin 300457, China;2. Tianjin Key Laboratory of Industrial Microbiology, Tianjin 300457, China;3. National Engineering Laboratory for Industrial Enzymes, Tianjin 300457, China;4. The College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China |
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| Abstract: | Microbial secretory phospholipases A2 (sPLA2s) are among the last discovered and least known members of this functionally diverse family of enzymes. We analyzed here two sPLA2s, named sPlaA and sPlaB, of the filamentous ascomycete Aspergillus oryzae. sPlaA and sPlaB consist of 222 and 160 amino acids, respectively, and share the conserved Cys and catalytic His-Asp residues typical of microbial sPLA2s. Two sPLA2s differ in pH optimum, Ca2+ requirement and expression profile. The splaA mRNA was strongly upregulated in response to carbon starvation, oxidative stress and during conidiation, while splaB was constitutively expressed at low levels and was weakly upregulated by heat shock. Experiments with sPLA2 overexpressing strains demonstrated that two enzymes produce subtly different phospholipid composition variations and also differ in their subcellular localization: sPlaA is most abundant in hyphal tips and secreted to the medium, whereas sPlaB predominantly localizes to the ER-like intracellular compartment. Both sPLA2 overexpressing strains were defective in conidiation, which was more pronounced for sPlaB overexpressors. Although no major morphological abnormality was detected in either ΔsplaA or ΔsplaB mutants, hyphal growth of ΔsplaB, but not that of ΔsplaA, displayed increased sensitivity to H2O2 treatment. These data indicate that two A. oryzae sPLA2 enzymes display distinct, presumably non-redundant, physiological functions. |
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