Novel bifunctional alpha-L-arabinofuranosidase/xylobiohydrolase (ABF3) from Penicillium purpurogenum.
The soft rot fungus Penicillium purpurogenum grows on a variety of natural substrates and secretes various isoforms of xylanolytic enzymes, including three arabinofuranosidases. This work describes the biochemical properties as well as the nucleotide and amino acid sequences of arabinofuranosidase 3 (ABF3). This enzyme has been purified to homogeneity. It is a glycosylated monomer with a molecular weight of 50,700 and can bind cellulose. The enzyme is active with p-nitrophenyl alpha-L-arabinofuranoside and p-nitrophenyl beta-D-xylopyranoside with a K(m) of 0.65 mM and 12 mM, respectively. The enzyme is active on xylooligosaccharides, yielding products of shorter length, including xylose. However, it does not hydrolyze arabinooligosaccharides. When assayed with polymeric substrates, little arabinose is liberated from arabinan and debranched arabinan; however, it hydrolyzes arabinose and releases xylooligosaccharides from arabinoxylan. Sequencing both ABF3 cDNA and genomic DNA reveals that this gene does not contain introns and that the open reading frame is 1,380 nucleotides in length. The deduced mature protein is composed of 433 amino acids residues and has a calculated molecular weight of 47,305. The deduced amino acid sequence has been validated by mass spectrometry analysis of peptides from purified ABF3. A total of 482 bp of the promoter were sequenced; putative binding sites for transcription factors such as CreA (four), XlnR (one), and AreA (three) and two CCAAT boxes were found. The enzyme has two domains, one similar to proteins of glycosyl hydrolase family 43 at the amino-terminal end and a family 6 carbohydrate binding module at the carboxyl end. ABF3 is the first described modular family 43 enzyme from a fungal source, having both alpha-L-arabinofuranosidase and xylobiohydrolase functionalities.
Ravanal
María Cristina
MC
Departamento de Ciencias Biológicas, Universidad Andrés Bello, República 217, Santiago, Chile.
Callegari
Eduardo
E
Eyzaguirre
Jaime
J
D001089:Arabinose / Q000031:analogs & derivatives / Q000378:metabolism; D002482:Cellulose / Q000378:metabolism; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D006027:Glycosides / Q000378:metabolism; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009844:Oligosaccharides / Q000378:metabolism; D016366:Open Reading Frames; D010407:Penicillium / Q000201:enzymology* / Q000235:genetics; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017422:Sequence Analysis, DNA; D014995:Xylosidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D004271:DNA, Fungal; D005656:Fungal Proteins; D006027:Glycosides; D009844:Oligosaccharides; C008646:4-nitrophenyl beta-D-xyloside; C017567:4-nitrophenyl-alpha-L-arabinofuranoside; D002482:Cellulose; D001089:Arabinose; D006026:Glycoside Hydrolases; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase; C019713:alpha-N-arabinofuranosidase