Identification and Characterization of a Novel Galactofuranose-Specific β-D-Galactofuranosidase from Streptomyces Species

RESEARCH ARTICLE Identification and Characterization of a Novel Galactofuranose-Specific β-DGalactofuranosidase from Streptomyces Species Emiko Matsunaga1, Yujiro Higuchi1, Kazuki Mori1, Nao Yairo1, Takuji Oka2, Saki Shinozuka3, Kosuke Tashiro1, Minoru Izumi3, Satoru Kuhara1, Kaoru Takegawa1* 1 Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 6–10–1 Hakozaki, Fukuoka, Japan, 2 Department of Applied Microbial Technology, Faculty of Biotechnology and Life Science, Sojo University, Kumamoto, Japan, 3 Graduate School of Environmental and Life Science, Okayama University, Okayama, Japan * OPEN ACCESS Citation: Matsunaga E, Higuchi Y, Mori K, Yairo N, Oka T, Shinozuka S, et al. (2015) Identification and Characterization of a Novel Galactofuranose-Specific β-D-Galactofuranosidase from Streptomyces Species. PLoS ONE 10(9): e0137230. doi:10.1371/ journal.pone.0137230 Editor: Olaf Kniemeyer, Hans-Knoell-Institute (HKI), GERMANY Received: June 23, 2015 Accepted: August 14, 2015 Published: September 4, 2015 Copyright: © 2015 Matsunaga et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract β-D-galactofuranose (Galf) is a component of polysaccharides and glycoconjugates and its transferase has been well analyzed. However, no β-D-galactofuranosidase (Galf-ase) gene has been identified in any organism. To search for a Galf-ase gene we screened soil samples and discovered a strain, identified as a Streptomyces species by the 16S ribosomal RNA gene analysis, that exhibits Galf-ase activity for 4-nitrophenyl β-D-galactofuranoside (pNP-β-D-Galf) in culture supernatants. By draft genome sequencing of the strain, named JHA19, we found four candidate genes encoding Galf-ases. Using recombinant proteins expressed in Escherichia coli, we found that three out of four candidates displayed the activity of not only Galf-ase but also α-L-arabinofuranosidase (Araf-ase), whereas the other one showed only the Galf-ase activity. This novel Galf-specific hydrolase is encoded by ORF1110 and has an optimum pH of 5.5 and a Km of 4.4 mM for the substrate pNP-β-DGalf. In addition, this enzyme was able to release galactose residue from galactomannan prepared from the filamentous fungus Aspergillus fumigatus, suggesting that natural polysaccharides could be also substrates. By the BLAST search using the amino acid sequence of ORF1110 Galf-ase, we found that there are homolog genes in both prokaryotes and eukaryotes, indicating that Galf-specific Galf-ases widely exist in microorganisms. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by JSPS KAKENHI (https://www.jsps.go.jp/english/index.html) Grant Numbers 26292054 to K. Takegawa and 26892022 to YH. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Introduction β-D-galactofuranose (Galf) is a constituent of polysaccharides and glycoconjugates that are present on the surface of the cell wall in many pathogenic bacteria and eukaryotes [1,2]. Galf is present in bacteria, filamentous fungi, trypanosomatids and nematodes, but not in yeasts nor in mammals [3,4]. Because Galf is known to be immunogenic to mammals [5–8], it is now a target molecule for anti-fungal reagents to suppress pathogenicity [1,2,4,9,10]. PLOS ONE | DOI:10.1371/journal.pone.0137230 September 4, 2015 1 / 16 Galactofuranose-Specific β-D-Galactofuranosidase In certain filamentous fungi, Galf is found in galactomannan (GM), galactomannoproteins modified with N-glycans and O-glycans and glycolipids [1,2,11–16]. Filamentous fungi enzymes involved in Galf-containing oligosaccharide synthesis have been well studied, especially in Aspergillus. For instance, in the model filamentous fungus Aspergillus nidulans, at the initial step of Galf-sugar chain synthesis, UDP-glucose, which is a donor substrate for α- and β-glucan synthesis, is converted to UDP-galactopyranose (UDP-Galp) by the UDP-glucose4-epimerase UgeA [17]. Then, UDP-Galp is converted to UDP-Galf by the UDP-Galf mutase UgmA (GlfA in Aspergillus fumigatus) [18–20]. These reactions occur in the cytoplasm. UDP-Galf is subsequently transported into the Golgi lumen by the UDP-Galf transporter UgtA (GlfB in A. fumigatus) which localized in the Golgi membrane [21,22]. To identify a Galf transferase gene in A. nidulans, we previously conducted reverse-genetics and biochemical approaches. We identified a gene named gfsA that encodes the Galf transferase localized to Golgi which function is to attach UDP-Galf onto the O-glycan chain [23]. ΔugmA, ΔugtA and ΔgfsA strains exhibit retarded hyphal morphology, suggesting that the Galf biosynthetic pathway is crucial for cell growth [19,22,23]. While the molecular mechanisms of the biosynthesis of Galf-containing sugar chains have been analyzed, enzymes involved in degradation and metabolism of Galf-oligosaccharides are not well known [1]. One such enzymes is β-D-galactofuranosidase (Galf-ase), which can release Galf from polysaccharides and glycoconjugates. There are reports about the purification of exoand endo-Galf-ases from the culture supernatant of several microorganisms [24–29]. However, no Galf-ase gene has been identified. α-L-arabinofuranosidase (Araf-ase), which hydrolyzes α-Larabinofuranoside (Araf), is structurally similar to Galf, and Araf-ase-encoding genes have been identified in Aspergillus species [10,30–37]. In Aspergillus niger, Araf-ases, which belong to glycosyl hydrolase family 51 (GH51) and 54 (GH54), have both Araf-ase and Galf-ase activities [30]. However, no gene encoding a Galf-ase-specific enzyme has been reported yet [38]. In this study, we screened soil samples for microorganisms that exhibit Galf-ase activity. The screen allowed us to identify a novel gene that encodes a Galf-ase-specific enzyme, which does not exhibit any Araf-ase activity. Materials and Methods Microorganism, cultivation and microscopy Bacteria were isolated from soil in Kagawa University, Japan. Since the area of the university is public, no specific permission was required to collect samples that did not include any endangered nor protected species. The isolated strain JHA19 (material number, QM2015–0042) has been deposited in the Material Management Center (MMC; http://mmc-u.jp/en/). YMG medium (0.4% yeast extract, 1% malt extract, 0.4% glucose and 2% agar, pH 7.3) was used for bacterial growth on plates and in liquid cultures, which were performed at 30°C with shaking at 200 rpm. Cells of the isolated strain JHA19 were cultured in YMG liquid medium for 3 days and observed under an Eclipse 80i microscope (Nikon) with a Pl (...truncated)