title: Substrate specificity and gene expression of two Penicillium chrysogenum α-L-arabinofuranosidases (AFQ1 and AFS1) belonging to glycoside hydrolase families 51 and 54.
abstract: We previously isolated two α-L-arabinofuranosidases (ABFs), termed AFQ1 and AFS1, from the culture filtrate of Penicillium chrysogenum 31B. afq1 and afs1 complementary DNAs encoding AFQ1 and AFS1 were isolated by in vitro cloning. The deduced amino acid sequences of AFQ1 and AFS1 are highly similar to those of Penicillium purpurogenum ABF 2 and ABF 1, respectively, which belong to glycoside hydrolase (GH) families 51 and 54, respectively. Pfam analysis revealed an "Alpha-L-AF_C" domain in AFQ1 and "ArabFuran-catal" and "AbfB" domains in AFS1. Semi-quantitative RT-PCR analysis indicated that the afq1 gene was constitutively expressed in P. chrysogenum 31B at a low level, although the expression was slightly induced with arabinose, arabinitol, arabinan, and arabinoxylan. In contrast, expression of the afs1 gene was strongly expressed by the above four carbohydrates and less strongly induced by galactan. Recombinant enzymes (rAFQ1 and rAFS1) expressed in Escherichia coli were active against both p-nitrophenyl α-L-arabinofuranoside and polysaccharides with different specificities. (1)H-NMR analysis revealed that rAFS1 degraded arabinofuranosyl side chains that were both singly and doubly linked to the backbones of arabinoxylan and L-arabinan. On the other hand, rAFQ1 preferentially released arabinose linked to C-3 of single-substituted xylose or arabinose residues in the two polysaccharides.
authors.affiliation: Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Osaka, 599-8531, Japan. sakamoto@biochem.osakafu-u.ac.jp
authors.forename: Tatsuji
authors.initials: T
authors.lastname: Sakamoto
authors.forename: Misako
authors.initials: M
authors.lastname: Inui
authors.forename: Kana
authors.initials: K
authors.lastname: Yasui
authors.forename: Sachiko
authors.initials: S
authors.lastname: Hosokawa
authors.forename: Hideshi
authors.initials: H
authors.lastname: Ihara
mesh_terms: D050260:Carbohydrate Metabolism; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D020869:Gene Expression Profiling; D015971:Gene Expression Regulation, Enzymologic; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010408:Penicillium chrysogenum / Q000201:enzymology* / Q000235:genetics; D017434:Protein Structure, Tertiary; D060888:Real-Time Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D004271:DNA, Fungal; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C019713:alpha-N-arabinofuranosidase