SBNet - Research Reports 1998

Inès Muñoz

PI: Ståhlberg (SLU)

Project: Structural studies of cellulases and other enzymes with potential for use in the cellulose industry and bio-organic synthesis


We have recently determined the molecular structure of the catalytic domain of cellobiohydrolase 58 (431 residues) at 2.1Å resolution by the molecular replacement method using the structure of T. reesei CBH 1 as search model. CBH 58 is the major cellulase produced by the white-rot fungus Phanerochaete chrysosporium , is classified into family 7 of the glycosyl hydrolases and it catalyses the hydrolysis with a net retention of the anomeric configuration. Its central structural motif is a large, mainly anti-parallel ß-sandwich that consist in two ß-sheets that pack face-to-face. The structure of the catalytic domain reveals a long cellulose-binding tunnel (~50 Å) that could provide a large number of interactions for a single cellulose chain. This tunnel is formed by loops on one face of the sandwich. CBH 58 is very similar to CBH 1 from Trichoderma reesei, but has a more exposed binding cleft due to deletions and other mutations in the loop regions [unpublished]. A less restricted active site is probably the reason why CBH 58 has a higher catalytic rate than CBH 1. Recent structures of catalytic deficient mutants of CBH 1 in complex with oligosaccharides showed that it binds 10 glycosyl units (subsites -7 to +3) and the cellulose chain is twisted around 180deg. along the enzyme. The structure lend further support to the hypothesis that CBH 1 acts processively from the reducing towards the non-reducing end along a cellulose chain. Superimposing the cellulose chain, as bound in CBH 1, on the structures of CBH 58 and T. reesei EG 1 show that the chain fit well in both enzymes without steric hindrance. CBH 58 is very similar to CBH 1 along the whole chain with all but three of the numerous interactions found in CBH 1 being present indicating that their modes of action on cellulose are very similar. In EG 1 the subsites flanking the point of cleavage are essentially preserved, but going towards the ends of the cleft corresponding interacting residues were not found in the same positions indicating that the cellulose chain may bind in a different configuration in EG 1. Probably it does not make the 180deg. twist. It seems rather unlikely that an endoglucanase would make such a twist internally in the cellulose chain prior to cleavage.

Now that CBH58 structure is done I will concentrate on endoglucanases. I have been working three weeks at VTT in Finland starting purification of an endoglucanase (EG 5) produced by T. reesei, and expect to get around 400 mg of protein for crystallisation. I have also received pure endoglucanase 2 (EG 2) from T. reesei and two homologous enzymes, EG44 and EG38 from P. chrysosporium and started crystallisation screening.


No publications. But I have started to prepare the manuscript describing the CBH 58 structure, and have presented the structure in invited lectures (2) and in a poster (3) at the following conferences/workshops:

1) Second Annual Conference of the Swedish Structural biology Network. Tällberg. May 98, 3 days. POSTER.

2) The roll of the cell wall degrading enzymes of Trichoderma harziarum in biocontrol. Tynset, Norway. July 31 - Aug 3, 1998. INVITED LECTURE.

3) Centre for Forest-biotechnology and Chemistry Midterm Meeting. Umeå. Nov. 9-13, 1998 POSTER.

4) Biotechnology Havana 98. Havana, Cuba. Nov 16-20, 1998. POSTER.

5) Avances en Biologia Molecular por jovenes investigadores en el extranjero. VI Workshop Sesion de Biologia Estructural. Madrid, Spain. Dec 29, 1998. INVITED LECTURE.

SBNet Latest update at 18 February, 1999.