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Active Site Loop Conformation Regulates Promiscuous Activity in a Lactonase from Geobacillus kaustophilus HTA426

Enzyme promiscuity is a prerequisite for fast divergent evolution of biocatalysts. A phosphotriesterase-like lactonase (PLL) from Geobacillus kaustophilus HTA426 (GkaP) exhibits main lactonase and promiscuous phosphotriesterase activities. To understand its catalytic and evolutionary mechanisms, we...

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Autores principales: Zhang, Yu, An, Jiao, Yang, Guang-Yu, Bai, Aixi, Zheng, Baisong, Lou, Zhiyong, Wu, Geng, Ye, Wei, Chen, Hai-Feng, Feng, Yan, Manco, Giuseppe
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4338136/
https://www.ncbi.nlm.nih.gov/pubmed/25706379
http://dx.doi.org/10.1371/journal.pone.0115130
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author Zhang, Yu
An, Jiao
Yang, Guang-Yu
Bai, Aixi
Zheng, Baisong
Lou, Zhiyong
Wu, Geng
Ye, Wei
Chen, Hai-Feng
Feng, Yan
Manco, Giuseppe
author_facet Zhang, Yu
An, Jiao
Yang, Guang-Yu
Bai, Aixi
Zheng, Baisong
Lou, Zhiyong
Wu, Geng
Ye, Wei
Chen, Hai-Feng
Feng, Yan
Manco, Giuseppe
author_sort Zhang, Yu
collection PubMed
description Enzyme promiscuity is a prerequisite for fast divergent evolution of biocatalysts. A phosphotriesterase-like lactonase (PLL) from Geobacillus kaustophilus HTA426 (GkaP) exhibits main lactonase and promiscuous phosphotriesterase activities. To understand its catalytic and evolutionary mechanisms, we investigated a “hot spot” in the active site by saturation mutagenesis as well as X-ray crystallographic analyses. We found that position 99 in the active site was involved in substrate discrimination. One mutant, Y99L, exhibited 11-fold improvement over wild-type in reactivity (k(cat)/K(m)) toward the phosphotriesterase substrate ethyl-paraoxon, but showed 15-fold decrease toward the lactonase substrate δ-decanolactone, resulting in a 157-fold inversion of the substrate specificity. Structural analysis of Y99L revealed that the mutation causes a ∼6.6 Å outward shift of adjacent loop 7, which may cause increased flexibility of the active site and facilitate accommodation and/or catalysis of organophosphate substrate. This study provides for the PLL family an example of how the evolutionary route from promiscuity to specificity can derive from very few mutations, which promotes alteration in the conformational adjustment of the active site loops, in turn draws the capacity of substrate binding and activity.
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spelling pubmed-43381362015-03-04 Active Site Loop Conformation Regulates Promiscuous Activity in a Lactonase from Geobacillus kaustophilus HTA426 Zhang, Yu An, Jiao Yang, Guang-Yu Bai, Aixi Zheng, Baisong Lou, Zhiyong Wu, Geng Ye, Wei Chen, Hai-Feng Feng, Yan Manco, Giuseppe PLoS One Research Article Enzyme promiscuity is a prerequisite for fast divergent evolution of biocatalysts. A phosphotriesterase-like lactonase (PLL) from Geobacillus kaustophilus HTA426 (GkaP) exhibits main lactonase and promiscuous phosphotriesterase activities. To understand its catalytic and evolutionary mechanisms, we investigated a “hot spot” in the active site by saturation mutagenesis as well as X-ray crystallographic analyses. We found that position 99 in the active site was involved in substrate discrimination. One mutant, Y99L, exhibited 11-fold improvement over wild-type in reactivity (k(cat)/K(m)) toward the phosphotriesterase substrate ethyl-paraoxon, but showed 15-fold decrease toward the lactonase substrate δ-decanolactone, resulting in a 157-fold inversion of the substrate specificity. Structural analysis of Y99L revealed that the mutation causes a ∼6.6 Å outward shift of adjacent loop 7, which may cause increased flexibility of the active site and facilitate accommodation and/or catalysis of organophosphate substrate. This study provides for the PLL family an example of how the evolutionary route from promiscuity to specificity can derive from very few mutations, which promotes alteration in the conformational adjustment of the active site loops, in turn draws the capacity of substrate binding and activity. Public Library of Science 2015-02-23 /pmc/articles/PMC4338136/ /pubmed/25706379 http://dx.doi.org/10.1371/journal.pone.0115130 Text en © 2015 Zhang et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Zhang, Yu
An, Jiao
Yang, Guang-Yu
Bai, Aixi
Zheng, Baisong
Lou, Zhiyong
Wu, Geng
Ye, Wei
Chen, Hai-Feng
Feng, Yan
Manco, Giuseppe
Active Site Loop Conformation Regulates Promiscuous Activity in a Lactonase from Geobacillus kaustophilus HTA426
title Active Site Loop Conformation Regulates Promiscuous Activity in a Lactonase from Geobacillus kaustophilus HTA426
title_full Active Site Loop Conformation Regulates Promiscuous Activity in a Lactonase from Geobacillus kaustophilus HTA426
title_fullStr Active Site Loop Conformation Regulates Promiscuous Activity in a Lactonase from Geobacillus kaustophilus HTA426
title_full_unstemmed Active Site Loop Conformation Regulates Promiscuous Activity in a Lactonase from Geobacillus kaustophilus HTA426
title_short Active Site Loop Conformation Regulates Promiscuous Activity in a Lactonase from Geobacillus kaustophilus HTA426
title_sort active site loop conformation regulates promiscuous activity in a lactonase from geobacillus kaustophilus hta426
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4338136/
https://www.ncbi.nlm.nih.gov/pubmed/25706379
http://dx.doi.org/10.1371/journal.pone.0115130
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