Molecular determinants of substrate and inhibitor specificities of the Penicillium griseofulvum family 11 xylanases.
Penicillium griseofulvum possesses two endo-(1,4)-beta-xylanase genes, PgXynA and PgXynB, belonging to family 11 glycoside hydrolases. The enzymes share 69% identity, a similar hydrolysis profile i.e. the predominant production of xylobiose and xylotriose as end products from wheat arabinoxylan and a specificity region of six potential xylose subsites, but differ in terms of catalytic efficiency which can be explained by subtle structural differences in the positioning of xylohexaose in the PgXynB model. Site-directed mutagenesis of the "thumb" region revealed structural basis of PgXynB substrate and inhibitor specificities. We produced variants displaying increased catalytic efficiency towards wheat arabinoxylan and xylo-oligosaccharides and identified specific determinants in PgXynB "thumb" region responsible for resistance to the wheat xylanase inhibitor XIP-I. Based on kinetic analysis and homology modeling, we suggested that Pro130(PgXynB), Lys131(PgXynB) and Lys132(PgXynB) hamper flexibility of the loop forming the "thumb" and interfere by steric hindrance with the inhibitor.
Cervera Tison
Marine
M
Biosciences ISM(2) UMR-CNRS-6263, Université Paul Cézanne Aix Marseille III, Av. Escadrille Normandie-Niemen, 13397 Marseille Cedex 20, France.
André-Leroux
Gwénaëlle
G
Lafond
Mickaël
M
Georis
Jacques
J
Juge
Nathalie
N
Berrin
Jean-Guy
JG
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000494:pharmacology; D043364:Endo-1,4-beta Xylanases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010407:Penicillium / Q000201:enzymology*; D010940:Plant Proteins / Q000494:pharmacology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity / Q000235:genetics
D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D010940:Plant Proteins; C485063:XIP-I protein, Triticum aestivum; D043364:Endo-1,4-beta Xylanases