ALTERATION OF THE CLEAVAGE MODE AND OF THE TRANSGLYCOSYLATION REACTIONS OF THE XYLANASE-A OF STREPTOMYCES-LIVIDANS-1326 BY SITE-DIRECTED MUTAGENESIS OF THE ASN173 RESIDUE

The amino acid replacement of Asn173 by Asp in the xylanase A (Xln A) of Streptomyces lividans significantly altered its enzymic properties. A time-course hydrolysis of xylan showed that the altered xylanase ([ N173D] Xln A) initially produced larger amounts of xylose (X(1)), xylo biose (X(2)) and xylotriose (X(3)) than Xln A, but less xylotetraose ( X(4)). The bond-cleavage frequencies were determined for both enzymes using xylopentaose (X(5)), xylotetraose (X(4)) and xylotriose (X(3)) l abelled at the reducing end of the molecule. Xln A hydrolysed X(5), yi elding 56% X(2) and 44% X(3), while [N173D]Xln A liberated 90% X(2) an d only 10% X(3). Both enzymes hydrolysed X(4) into 100% X(2) and X(3) into 100% X(1). Transglycosylation reactions were detected in HPLC hyd rolysis patterns using high substrate concentrations, where larger pro ducts than the starting substrates were formed. Their subsequent degra dation also affected the yield of hydrolysis products. Using X(5) as s ubstrate, products from xylohexaose (X(6)) up to xylooligosides larger than xylooctaose (X(8)) were synthesized by Xln A, while [N173D]Xln A produced only a small amount of xyloheptaose (X(7)) and X(8). Xln A h ydrolysed X(5) into an equivalent amount of X(4) and X(2) and 1.5 fold more X(3). However, [N173D]Xln A yielded the same amount of X(3) and X(2) but half as much X(4). With X(4) as substrate, Xln A synthesized twofold more X(7) and X(6) than [N173D]Xln A. Xln A liberated 1.4-fold more X(3) than X(2), while [N173D]Xln. A yielded twofold more X(2) th an X(3). Xln A liberated almost fourfold more X(2) than X(1) from X(3) , while [N173D]Xln A produced only twofold more X(2) than X(1). These results indicated that the negative charge introduced by the mutation greatly affected the transglycosylation reactions catalysed by this xy lanase.