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Structure-oriented substrate specificity engineering of aldehyde-deformylating oxygenase towards aldehydes carbon chain length

BACKGROUND: Aldehyde-deformylating oxygenase (ADO) is an important enzyme involved in the biosynthetic pathway of fatty alk(a/e)nes in cyanobacteria. However, ADO exhibits quite low chain-length specificity with respect to the substrates ranging from C(4) to C(18) aldehydes, which is not suitable fo...

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Autores principales: Bao, Luyao, Li, Jian-Jun, Jia, Chenjun, Li, Mei, Lu, Xuefeng
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5007808/
https://www.ncbi.nlm.nih.gov/pubmed/27588038
http://dx.doi.org/10.1186/s13068-016-0596-9
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author Bao, Luyao
Li, Jian-Jun
Jia, Chenjun
Li, Mei
Lu, Xuefeng
author_facet Bao, Luyao
Li, Jian-Jun
Jia, Chenjun
Li, Mei
Lu, Xuefeng
author_sort Bao, Luyao
collection PubMed
description BACKGROUND: Aldehyde-deformylating oxygenase (ADO) is an important enzyme involved in the biosynthetic pathway of fatty alk(a/e)nes in cyanobacteria. However, ADO exhibits quite low chain-length specificity with respect to the substrates ranging from C(4) to C(18) aldehydes, which is not suitable for producing fuels with different properties or different chain lengths. RESULTS: Based on the crystal structures of cADOs (cyanobacterial ADO) with substrate analogs bound, some amino acids affecting the substrate specificity of cADO were identified, including the amino acids close to the aldehyde group and the hydrophobic tail of the substrate and those along the substrate channel. Using site-directed mutagenesis, selected amino acids were replaced with bulky ones introducing steric hindrance to the binding pocket via large functional groups. All mutants were overexpressed, purified and kinetically characterized. All mutants, except F87Y, displayed dramatically reduced activity towards C(14,16,18) aldehydes. Notably, the substrate preferences of some mutants towards different chain-length substrates were enhanced: I24Y for n-heptanal, I27F for n-decanal and n-dodecanal, V28F for n-dodecanal, F87Y for n-decanal, C70F for n-hexanal, A118F for n-butanal, A121F for C(4,6,7) aldehydes, V184F for n-dodecanal and n-decanal, M193Y for C(6–10) aldehydes and L198F for C(7–10) aldehydes. The impact of the engineered cADO mutants on the change of the hydrocarbon profile was demonstrated by co-expressing acyl-ACP thioesterase BTE, fadD and V184F in E. coli, showing that n-undecane was the main fatty alkane. CONCLUSIONS: Some amino acids, which can control the chain-length selectivity of substrates of cADO, were identified. The substrate specificities of cADO were successfully changed through structure-guided protein engineering, and some mutants displayed different chain-length preference. The in vivo experiments of V184F in genetically engineered E. coli proved the importance of engineered cADOs on the distribution of the fatty alkane profile. The results would be helpful for the production of fatty alk(a/e)nes in cyanobacteria with different properties. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13068-016-0596-9) contains supplementary material, which is available to authorized users.
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spelling pubmed-50078082016-09-02 Structure-oriented substrate specificity engineering of aldehyde-deformylating oxygenase towards aldehydes carbon chain length Bao, Luyao Li, Jian-Jun Jia, Chenjun Li, Mei Lu, Xuefeng Biotechnol Biofuels Research BACKGROUND: Aldehyde-deformylating oxygenase (ADO) is an important enzyme involved in the biosynthetic pathway of fatty alk(a/e)nes in cyanobacteria. However, ADO exhibits quite low chain-length specificity with respect to the substrates ranging from C(4) to C(18) aldehydes, which is not suitable for producing fuels with different properties or different chain lengths. RESULTS: Based on the crystal structures of cADOs (cyanobacterial ADO) with substrate analogs bound, some amino acids affecting the substrate specificity of cADO were identified, including the amino acids close to the aldehyde group and the hydrophobic tail of the substrate and those along the substrate channel. Using site-directed mutagenesis, selected amino acids were replaced with bulky ones introducing steric hindrance to the binding pocket via large functional groups. All mutants were overexpressed, purified and kinetically characterized. All mutants, except F87Y, displayed dramatically reduced activity towards C(14,16,18) aldehydes. Notably, the substrate preferences of some mutants towards different chain-length substrates were enhanced: I24Y for n-heptanal, I27F for n-decanal and n-dodecanal, V28F for n-dodecanal, F87Y for n-decanal, C70F for n-hexanal, A118F for n-butanal, A121F for C(4,6,7) aldehydes, V184F for n-dodecanal and n-decanal, M193Y for C(6–10) aldehydes and L198F for C(7–10) aldehydes. The impact of the engineered cADO mutants on the change of the hydrocarbon profile was demonstrated by co-expressing acyl-ACP thioesterase BTE, fadD and V184F in E. coli, showing that n-undecane was the main fatty alkane. CONCLUSIONS: Some amino acids, which can control the chain-length selectivity of substrates of cADO, were identified. The substrate specificities of cADO were successfully changed through structure-guided protein engineering, and some mutants displayed different chain-length preference. The in vivo experiments of V184F in genetically engineered E. coli proved the importance of engineered cADOs on the distribution of the fatty alkane profile. The results would be helpful for the production of fatty alk(a/e)nes in cyanobacteria with different properties. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13068-016-0596-9) contains supplementary material, which is available to authorized users. BioMed Central 2016-08-31 /pmc/articles/PMC5007808/ /pubmed/27588038 http://dx.doi.org/10.1186/s13068-016-0596-9 Text en © The Author(s) 2016 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research
Bao, Luyao
Li, Jian-Jun
Jia, Chenjun
Li, Mei
Lu, Xuefeng
Structure-oriented substrate specificity engineering of aldehyde-deformylating oxygenase towards aldehydes carbon chain length
title Structure-oriented substrate specificity engineering of aldehyde-deformylating oxygenase towards aldehydes carbon chain length
title_full Structure-oriented substrate specificity engineering of aldehyde-deformylating oxygenase towards aldehydes carbon chain length
title_fullStr Structure-oriented substrate specificity engineering of aldehyde-deformylating oxygenase towards aldehydes carbon chain length
title_full_unstemmed Structure-oriented substrate specificity engineering of aldehyde-deformylating oxygenase towards aldehydes carbon chain length
title_short Structure-oriented substrate specificity engineering of aldehyde-deformylating oxygenase towards aldehydes carbon chain length
title_sort structure-oriented substrate specificity engineering of aldehyde-deformylating oxygenase towards aldehydes carbon chain length
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5007808/
https://www.ncbi.nlm.nih.gov/pubmed/27588038
http://dx.doi.org/10.1186/s13068-016-0596-9
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