Identification of two GH27 bifunctional proteins with beta-L-arabinopyranosidase/alpha-D-galactopyranosidase activities from Fusarium oxysporum.
Two distinct extracellular bifunctional proteins with beta-L-arabinopyranosidase/alpha-D-galactopyranosidase activities were purified from the culture filtrate of Fusarium oxysporum 12S. The molecular masses of the enzymes were estimated to be 55 (Fo/AP1) and 73 kDa (Fo/AP2) by SDS-PAGE. They hydrolyzed both p-nitrophenyl beta-L-arabinopyranoside and p-nitrophenyl alpha-D-galactopyranoside with different specificities. Fo/AP1 also showed low activity towards alpha-D-galactopyranosyl oligosaccharides such as raffinose. Interestingly, both enzymes hydrolyzed larch wood arabinogalactan (releasing arabinose) but not carob galactomannan, which has alpha-D-galactopyranosyl side chains. When larch wood arabinogalactan was incubated with excess Fo/AP1 or Fo/AP2, both enzymes released approximately 10% of the total arabinose in the substrate. cDNAs encoding Fo/AP1 and Fo/AP2 (Foap1 and Foap2) were isolated by in vitro cloning. The coding sequences of Foap1 and Foap2 genes were 1,647 and 1,620 bp in length and encode polypeptides of 549 and 540 amino acids, respectively. The N-terminal halves of both proteins had high similarity to putative conserved domains of the melibiase superfamily (Pfam account number 02065). The deduced amino acid sequences of the two enzymes indicate that they belong to glycosyl hydrolase family 27. Moreover, the C-terminal regions of both proteins contain a putative family 35 carbohydrate-binding module.
Sakamoto
Tatsuji
T
Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Osaka, Japan. sakamoto@biochem.osakafu-u.ac.jp
Tsujitani
Yuya
Y
Fukamachi
Keiko
K
Taniguchi
Yuya
Y
Ihara
Hideshi
H
D000595:Amino Acid Sequence; D001089:Arabinose / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004798:Enzymes / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D005670:Fusarium / Q000201:enzymology* / Q000235:genetics; D005690:Galactose / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009844:Oligosaccharides / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D018076:DNA, Complementary; D004271:DNA, Fungal; D004798:Enzymes; D005656:Fungal Proteins; D009844:Oligosaccharides; D001089:Arabinose; D005690:Galactose