The active site of <i>Trichoderma reesei</i> cellobiohydrolase II: the role of tyrosine 169

Koivula, Anu; Reinikainen, Tapani; Ruohonen, Laura; Valkeajärvi, Anne; Claeyssens, Marc; Teleman, Olle; Kleywegt, Gerard J.; Szardenings, Michael; Rouvinen, Juha; Jones, T. Alwyn; Teeri, Tuula T.

doi:10.1093/protein/9.8.691

Cited by 79 publications

(90 citation statements)

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“…In the case of the CBH II library, the high parent pair sequence identifies (82, 66, and 64%) suggest that only minor structure deviations are likely (Ͻ1 Å r.m.s.d.) (12). We can explicitly evaluate this possibility by comparing crystallographic structures for H. insolens and H. jecorina CBH II (C. thermophilum CBH II lacks a crystal structure but is 82% identical to H. insolens).…”

Section: Discussionmentioning

confidence: 99%

SCHEMA Recombination of a Fungal Cellulase Uncovers a Single Mutation That Contributes Markedly to Stability

Heinzelman

Snow

Smith

et al. 2009

Journal of Biological Chemistry

112

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A quantitative linear model accurately (R 2 ‫؍‬ 0.88) describes the thermostabilities of 54 characterized members of a family of fungal cellobiohydrolase class II (CBH II) cellulase chimeras made by SCHEMA recombination of three fungal enzymes, demonstrating that the contributions of SCHEMA sequence blocks to stability are predominantly additive. Thirty-one of 31 predicted thermostable CBH II chimeras have thermal inactivation temperatures higher than the most thermostable parent CBH II, from Humicola insolens, and the model predicts that hundreds more CBH II chimeras share this superior thermostability. Eight of eight thermostable chimeras assayed hydrolyze the solid cellulosic substrate Avicel at temperatures at least 5°C above the most stable parent, and seven of these showed superior activity in 16-h Avicel hydrolysis assays. The sequence-stability model identified a single block of sequence that adds 8.5°C to chimera thermostability. Mutating individual residues in this block identified the C313S substitution as responsible for the entire thermostabilizing effect. Introducing this mutation into the two recombination parent CBH IIs not featuring it (Hypocrea jecorina and H. insolens) decreased inactivation, increased maximum Avicel hydrolysis temperature, and improved long time hydrolysis performance. This mutation also stabilized and improved Avicel hydrolysis by Phanerochaete chrysosporium CBH II, which is only 55-56% identical to recombination parent CBH IIs. Furthermore, the C313S mutation increased total H. jecorina CBH II activity secreted by the Saccharomyces cerevisiae expression host more than 10-fold. Our results show that SCHEMA structure-guided recombination enables quantitative prediction of cellulase chimera thermostability and efficient identification of stabilizing mutations.SCHEMA is a computational approach to identifying blocks of sequence that minimize structural disruption when they are recombined in chimeric proteins (1). SCHEMA recombination of eight blocks from three fungal cellobiohydrolase class II (CBH II) 2 genes was used in our previous work to create a library of 3 8 ϭ 6,561 chimeric sequences, all having the native Hypocrea jecorina cellulose binding module and linker and observed to feature a degree of glycosylation similar to that found in native CBH IIs secreted by fungi (2). Synthesis and characterization of selected CBH II chimeras expressed in Saccharomyces cerevisiae revealed enzymes with thermostabilities and cellulose hydrolysis performance superior to those of the parent enzymes from Humicola insolens, H. jecorina, and Chaetomium thermophilum.Our prior analysis showed that a qualitative model based on sequence-stability data from 23 functional chimeras (categorizing blocks as destabilizing, stabilizing, or neutral) could identify highly stable chimeras in the SCHEMA library (2). When studying SCHEMA recombination of a bacterial cytochrome P450, we previously estimated that building a quantitative regression model would require stability measurements for at least 35 re...

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Section: Discussionmentioning

confidence: 99%

SCHEMA Recombination of a Fungal Cellulase Uncovers a Single Mutation That Contributes Markedly to Stability

Heinzelman

Snow

Smith

et al. 2009

Journal of Biological Chemistry

112

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“…The sequence encoding CBD CBH1 with a six-residue linker was cloned from pEMF5 (23). The segment encoding CBD CBH2 and six residues from the adjacent linker was carried by pTTc9 (24). The segment encoding CBD Cex and seven residues from the adjacent linker was cloned from pOxscFv-CBD (25).…”

Section: Methodsmentioning

confidence: 99%

Cellulose-binding domains promote hydrolysis of different sites on crystalline cellulose

Carrard

Koivula

Söderlund

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

242

180

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“…at least five different folds are known for cellulases (1cec, 1cb2, 1cel, 1clc, 2eng;Dominguez et al, 1995;Koivula et al, 1996;Divne et al, 1994; M. B. Lascombe, H. Souchon, M. Juy & P. M. Alzari, unpublished work; . CAZymes have a highly variable modular structure, with the catalytic module carrying a variable number of ancillary modules.…”

Section: Figurementioning

confidence: 99%

Viewing the human microbiome through three-dimensional glasses: integrating structural and functional studies to better define the properties of myriad carbohydrate-active enzymes

2010

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Recent studies have provided an unprecedented view of the trillions of microbes associated with the human body. The human microbiome harbors tremendous diversity at multiple levels: the species that colonize each individual and each body habitat; the genes that are found in each organism's genome; the expression of these genes and the interactions and activities of their protein products. The sources of this diversity are wide-ranging and reflect both environmental and host factors. A major challenge moving forward is defining the precise functions of members of various families of proteins represented in our microbiomes, including the highly diverse carbohydrate-active enzymes (CAZymes) involved in numerous biologically important chemical transformations, such as the degradation of complex dietary polysaccharides. Coupling metagenomic analyses to structural genomics initiatives and to biochemical and other functional assays of CAZymes will be essential for determining how these as well as other microbiome-encoded proteins operate to shape the properties of microbial communities and their human hosts.

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The active site of Trichoderma reesei cellobiohydrolase II: the role of tyrosine 169

Cited by 79 publications

References 1 publication

SCHEMA Recombination of a Fungal Cellulase Uncovers a Single Mutation That Contributes Markedly to Stability

SCHEMA Recombination of a Fungal Cellulase Uncovers a Single Mutation That Contributes Markedly to Stability

Cellulose-binding domains promote hydrolysis of different sites on crystalline cellulose

Viewing the human microbiome through three-dimensional glasses: integrating structural and functional studies to better define the properties of myriad carbohydrate-active enzymes

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