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Journal of Biochemistry Advance Access originally published online on March 23, 2007
Journal of Biochemistry 2007 141(5):709-717; doi:10.1093/jb/mvm072
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© 2007 The Japanese Biochemical Society.

Molecular Anatomy of the Alkaliphilic Xylanase from Bacillus halodurans C-125

Mamoru Nishimoto1, Shinya Fushinobu2, Akimasa Miyanaga2, Motomitsu Kitaoka1,* and Kiyoshi Hayashi1

1Enzyme Laboratory, National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642; and 2Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

* To whom correspondence should be addressed. Tel: +81-29-838-8071, Fax: +81-29-838-7321, E-mail: mkitaoka{at}affrc.go.jp

Received February 5, 2007; Accepted February 26, 2007


  Abstract

Two regions in xylanase A from Bacillus halodurans C-125 (XynA), an alkaliphilic xylanase, were identified to be responsible for its activity at basic pH by comparing the dissociation constants of the XynA proton donor Glu residue (pKe2 and pKes2) with those of xylanase B from Clostridium stercorarium F9 (XynB) and their mutants constructed by substituting either Ser137/Asn127 of XynA/XynB or the 4th loop, designed based on the structural difference close to the proton donor. The substitution of XynB at Asn127 into Ser increased pKe2 by 0.37. The effect is explained that the positive charge of His126 likely affects the proton donor via Asn127 and a water molecule in XynB, resulting in a decrease in pKe2, whereas such interactions were not observed with Ser. The substitution of XynB at the 4th loop into XynA (XynB Loop4A) increased the pKe2 and pKes2 values by 0.29 and 0.62, respectively. The effect of the 4th loop in XynA is likely due to a hydrogen bond between Asp199 in the loop and Tyr239, which interacts with both the proton donors Glu195 and Arg204, with flexibility of the loop. Both the mutations independently affected the increases in pKe2.

Key Words: family 10 xylanase, pH-activity relationship

Abbreviations: XynA, xylanase A from Bacillus halodurans C-125; XynB, xylanase B from Clostridium stercorarium F9; XynT6, xylanase T6 from Geobacillus stearothermophilus; pNP-X2, p-nitrophenyl xylobioside


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