caa a22 4500 605499403 CHVBK 20210128100550.0 cr unu---uuuuu 210128e20151001xx s 000 0 eng 10.1007/s00253-015-6600-7 doi (NATIONALLICENCE)springer-10.1007/s00253-015-6600-7 Molecular characterization of a Penicillium chrysogenum exo-rhamnogalacturonan lyase that is structurally distinct from other polysaccharide lyase family proteins [Elektronische Daten] [Marin Iwai, Takuya Kawakami, Takeshi Ikemoto, Daisuke Fujiwara, Shigeo Takenaka, Masami Nakazawa, Mitsuhiro Ueda, Tatsuji Sakamoto] We previously described an endo-acting rhamnogalacturonan (RG) lyase, termed PcRGL4A, of Penicillium chrysogenum 31B. Here, we describe a second RG lyase, called PcRGLX. We determined the cDNA sequence of the Pcrglx gene, which encodes PcRGLX. Based on analyses using a BLAST search and a conserved domain search, PcRGLX was found to be structurally distinct from known RG lyases and might belong to a new polysaccharide lyase family together with uncharacterized fungal proteins of Nectria haematococca, Aspergillus oryzae, and Fusarium oxysporum. The Pcrglx cDNA gene product (rPcRGLX) expressed in Escherichia coli demonstrated specific activity against RG but not against homogalacturonan. Divalent cations were not essential for the enzymatic activity of rPcRGLX. rPcRGLX mainly released unsaturated galacturonosyl rhamnose (ΔGR) from RG backbones used as the substrate from the initial stage of the reaction, indicating that the enzyme can be classified as an exo-acting RG lyase (EC 4.2.2.24). This is the first report of an RG lyase with this mode of action in Eukaryota. rPcRGLX acted synergistically with PcRGL4A to degrade soybean RG and released ΔGR. This ΔGR was partially decorated with galactose (Gal) residues, indicating that rPcRGLX preferred oligomeric RGs to polymeric RGs, that the enzyme did not require Gal decoration of RG backbones for degradation, and that the enzyme bypassed the Gal side chains of RG backbones. These characteristics of rPcRGLX might be useful in the determination of complex structures of pectins. Springer-Verlag Berlin Heidelberg, 2015 Exo-acting rhamnogalacturonan lyase nationallicence Nucleotide sequence nationallicence Overexpression nationallicence Penicillium chrysogenum nationallicence Unsaturated galacturonosyl rhamnose nationallicence Iwai Marin Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 599-8531, Sakai, Osaka, Japan aut Kawakami Takuya Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 599-8531, Sakai, Osaka, Japan aut Ikemoto Takeshi Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 599-8531, Sakai, Osaka, Japan aut Fujiwara Daisuke Department of Biological Science, Graduate School of Science, Osaka Prefecture University, 599-8531, Sakai, Osaka, Japan aut Takenaka Shigeo Division of Veterinary science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 598-8531, Izumisano, Osaka, Japan aut Nakazawa Masami Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 599-8531, Sakai, Osaka, Japan aut Ueda Mitsuhiro Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 599-8531, Sakai, Osaka, Japan aut Sakamoto Tatsuji Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 599-8531, Sakai, Osaka, Japan aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/20(2015-10-01), 8515-8525 0175-7598 99:20<8515 2015 99 253 https://doi.org/10.1007/s00253-015-6600-7 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6600-7 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Iwai Marin Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 599-8531, Sakai, Osaka, Japan aut NATIONALLICENCE 700 1- Kawakami Takuya Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 599-8531, Sakai, Osaka, Japan aut NATIONALLICENCE 700 1- Ikemoto Takeshi Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 599-8531, Sakai, Osaka, Japan aut NATIONALLICENCE 700 1- Fujiwara Daisuke Department of Biological Science, Graduate School of Science, Osaka Prefecture University, 599-8531, Sakai, Osaka, Japan aut NATIONALLICENCE 700 1- Takenaka Shigeo Division of Veterinary science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 598-8531, Izumisano, Osaka, Japan aut NATIONALLICENCE 700 1- Nakazawa Masami Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 599-8531, Sakai, Osaka, Japan aut NATIONALLICENCE 700 1- Ueda Mitsuhiro Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 599-8531, Sakai, Osaka, Japan aut NATIONALLICENCE 700 1- Sakamoto Tatsuji Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 599-8531, Sakai, Osaka, Japan aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/20(2015-10-01), 8515-8525 0175-7598 99:20<8515 2015 99 253