Characterization of abn2 (yxiA), encoding a Bacillus subtilis GH43 arabinanase, Abn2, and its role in arabino-polysaccharide degradation.
The extracellular depolymerization of arabinopolysaccharides by microorganisms is accomplished by arabinanases, xylanases, and galactanases. Here, we characterize a novel endo-alpha-1,5-l-arabinanase (EC 3.2.1.99) from Bacillus subtilis, encoded by the yxiA gene (herein renamed abn2) that contributes to arabinan degradation. Functional studies by mutational analysis showed that Abn2, together with previously characterized AbnA, is responsible for the majority of the extracellular arabinan activity in B. subtilis. Abn2 was overproduced in Escherichia coli, purified from the periplasmic fraction, and characterized with respect to substrate specificity and biochemical and physical properties. With linear-alpha-1,5-l-arabinan as the preferred substrate, the enzyme exhibited an apparent K(m) of 2.0 mg ml(-1) and V(max) of 0.25 mmol min(-1) mg(-1) at pH 7.0 and 50 degrees C. RNA studies revealed the monocistronic nature of abn2. Two potential transcriptional start sites were identified by primer extension analysis, and both a sigma(A)-dependent and a sigma(H)-dependent promoter were located. Transcriptional fusion studies revealed that the expression of abn2 is stimulated by arabinan and pectin and repressed by glucose; however, arabinose is not the natural inducer. Additionally, trans-acting factors and cis elements involved in transcription were investigated. Abn2 displayed a control mechanism at a level of gene expression different from that observed with AbnA. These distinct regulatory mechanisms exhibited by two members of extracellular glycoside hydrolase family 43 (GH43) suggest an adaptative strategy of B. subtilis for optimal degradation of arabinopolysaccharides.
Inácio
José Manuel
JM
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Avenida de República-EAN, 2780-157 Oeiras, Portugal.
de Sá-Nogueira
Isabel
I
D001089:Arabinose / Q000737:chemistry / Q000378:metabolism*; D001412:Bacillus subtilis / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D004252:DNA Mutational Analysis; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D005947:Glucose / Q000494:pharmacology; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010368:Pectins / Q000494:pharmacology; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D014158:Transcription, Genetic / Q000187:drug effects
D001426:Bacterial Proteins; D011134:Polysaccharides; D011994:Recombinant Proteins; C030080:araban; D010368:Pectins; D001089:Arabinose; D006026:Glycoside Hydrolases; C082526:arabinan endo-1,5-alpha-L-arabinosidase; D005947:Glucose