Structural redesign of lipase B from Candida antarctica by circular permutation and incremental truncation
Circular permutation of Candida antarctica lipase B yields several enzyme variants with substantially increased catalytic activity. To better understand the structural and functional consequences of protein termini reorganization, we have applied protein engineering and x-ray crystallography to cp28...
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ftpubmed:oai:pubmedcentral.nih.gov:2753740 2023-05-15T13:40:57+02:00 Structural redesign of lipase B from Candida antarctica by circular permutation and incremental truncation Qian, Zhen Horton, John R. Cheng, Xiaodong Lutz, Stefan 2009-08-13 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2753740 http://www.ncbi.nlm.nih.gov/pubmed/19683009 https://doi.org/10.1016/j.jmb.2009.08.008 en eng http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2753740 http://www.ncbi.nlm.nih.gov/pubmed/19683009 http://dx.doi.org/10.1016/j.jmb.2009.08.008 © 2009 Elsevier Ltd. All rights reserved. Article Text 2009 ftpubmed https://doi.org/10.1016/j.jmb.2009.08.008 2013-09-02T17:22:33Z Circular permutation of Candida antarctica lipase B yields several enzyme variants with substantially increased catalytic activity. To better understand the structural and functional consequences of protein termini reorganization, we have applied protein engineering and x-ray crystallography to cp283, one of the most active hydrolase variants. Our initial investigation has focused on the role of an extended surface loop, created by linking the native N and C-termini, on protein integrity. Incremental truncation of the loop partially compensates for observed losses in secondary structure and the permutants’ temperature of unfolding. Unexpectedly, the improvements are accompanied by quaternary structure changes from monomer to dimer. The crystal structures of one truncated variant (cp283Δ7) in the apo-form determined at 1.49Å resolution and with a bound phosphonate inhibitor at 1.69Å resolution confirmed the formation of a homodimer by swapping of the enzyme’s 35-residue N-terminal region. Separately, the new protein termini at amino acid positions 282/283 convert the narrow access tunnel to the catalytic triad into a broad crevice for accelerated substrate entry and product exit while preserving the native active site topology for optimal catalytic turnover. Text Antarc* Antarctica PubMed Central (PMC) Journal of Molecular Biology 393 1 191 201 |
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Article Qian, Zhen Horton, John R. Cheng, Xiaodong Lutz, Stefan Structural redesign of lipase B from Candida antarctica by circular permutation and incremental truncation |
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Article |
description |
Circular permutation of Candida antarctica lipase B yields several enzyme variants with substantially increased catalytic activity. To better understand the structural and functional consequences of protein termini reorganization, we have applied protein engineering and x-ray crystallography to cp283, one of the most active hydrolase variants. Our initial investigation has focused on the role of an extended surface loop, created by linking the native N and C-termini, on protein integrity. Incremental truncation of the loop partially compensates for observed losses in secondary structure and the permutants’ temperature of unfolding. Unexpectedly, the improvements are accompanied by quaternary structure changes from monomer to dimer. The crystal structures of one truncated variant (cp283Δ7) in the apo-form determined at 1.49Å resolution and with a bound phosphonate inhibitor at 1.69Å resolution confirmed the formation of a homodimer by swapping of the enzyme’s 35-residue N-terminal region. Separately, the new protein termini at amino acid positions 282/283 convert the narrow access tunnel to the catalytic triad into a broad crevice for accelerated substrate entry and product exit while preserving the native active site topology for optimal catalytic turnover. |
format |
Text |
author |
Qian, Zhen Horton, John R. Cheng, Xiaodong Lutz, Stefan |
author_facet |
Qian, Zhen Horton, John R. Cheng, Xiaodong Lutz, Stefan |
author_sort |
Qian, Zhen |
title |
Structural redesign of lipase B from Candida antarctica by circular permutation and incremental truncation |
title_short |
Structural redesign of lipase B from Candida antarctica by circular permutation and incremental truncation |
title_full |
Structural redesign of lipase B from Candida antarctica by circular permutation and incremental truncation |
title_fullStr |
Structural redesign of lipase B from Candida antarctica by circular permutation and incremental truncation |
title_full_unstemmed |
Structural redesign of lipase B from Candida antarctica by circular permutation and incremental truncation |
title_sort |
structural redesign of lipase b from candida antarctica by circular permutation and incremental truncation |
publishDate |
2009 |
url |
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2753740 http://www.ncbi.nlm.nih.gov/pubmed/19683009 https://doi.org/10.1016/j.jmb.2009.08.008 |
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Antarc* Antarctica |
genre_facet |
Antarc* Antarctica |
op_relation |
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2753740 http://www.ncbi.nlm.nih.gov/pubmed/19683009 http://dx.doi.org/10.1016/j.jmb.2009.08.008 |
op_rights |
© 2009 Elsevier Ltd. All rights reserved. |
op_doi |
https://doi.org/10.1016/j.jmb.2009.08.008 |
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Journal of Molecular Biology |
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393 |
container_issue |
1 |
container_start_page |
191 |
op_container_end_page |
201 |
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1766143653124767744 |