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Applied and Environmental Microbiology, May 2007, p. 3165-3172, Vol. 73, No. 10
0099-2240/07/$08.00+0     doi:10.1128/AEM.02960-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Processivity, Substrate Binding, and Mechanism of Cellulose Hydrolysis by Thermobifida fusca Cel9A{triangledown}

Yongchao Li,1 Diana C. Irwin,2 and David B. Wilson2*

Field of Microbiology, Cornell University, Ithaca, New York 14850,1 Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 148532

Received 20 December 2006/ Accepted 10 March 2007

Thermobifida fusca Cel9A-90 is a processive endoglucanase consisting of a family 9 catalytic domain (CD), a family 3c cellulose binding module (CBM3c), a fibronectin III-like domain, and a family 2 CBM. This enzyme has the highest activity of any individual T. fusca enzyme on crystalline substrates, particularly bacterial cellulose (BC). Mutations were introduced into the CD or the CBM3c of Cel9A-68 using site-directed mutagenesis. The mutant enzymes were expressed in Escherichia coli; purified; and tested for activity on four substrates, ligand binding, and processivity. The results show that H125 and Y206 play an important role in activity by forming a hydrogen bonding network with the catalytic base, D58; another important supporting residue, D55; and Glc(–1) O1. R378, a residue interacting with Glc(+1), plays an important role in processivity. Several enzymes with mutations in the subsites Glc(–2) to Glc(–4) had less than 15% activity on BC and markedly reduced processivity. Mutant enzymes with severalfold-higher activity on carboxymethyl cellulose (CMC) were found in the subsites from Glc(–2) to Glc(–4). The CBM3c mutant enzymes, Y520A, R557A/E559A, and R563A, had decreased activity on BC but had wild-type or improved processivity. Mutation of D513, a conserved residue at the end of the CBM, increased activity on crystalline cellulose. Previous work showed that deletion of the CBM3c abolished crystalline activity and processivity. This study shows that it is residues in the catalytic cleft that control processivity while the CBM3c is important for loose binding of the enzyme to the crystalline cellulose substrate.

* Corresponding author. Mailing address: 458 Biotechnology Building, Cornell University, Ithaca, NY 14850. Phone: (607) 255-5706. Fax: (607) 255-2428. E-mail: dbw3{at}cornell.edu

{triangledown} Published ahead of print on 16 March 2007.

Applied and Environmental Microbiology, May 2007, p. 3165-3172, Vol. 73, No. 10
0099-2240/07/$08.00+0     doi:10.1128/AEM.02960-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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