Biophysical and structural studies reveal marginal stability of a crucial hydrocarbon biosynthetic enzyme acyl ACP reductase

Abstract Acyl-ACP reductase (AAR) is one of the two key cyanobacterial enzymes along with aldehyde deformylating oxygenase (ADO) involved in the synthesis of long-chain alkanes, a drop-in biofuel. The enzyme is prone to aggregation when expressed in Escherichia coli, leading to varying alkane levels...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Ashima Sharma, Tabinda Shakeel, Mayank Gupta, Girish H. Rajacharya, Syed Shams Yazdani
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/f138d43b90734f25a7dca822c071ff85
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:f138d43b90734f25a7dca822c071ff85
record_format	dspace
spelling	oai:doaj.org-article:f138d43b90734f25a7dca822c071ff852021-12-02T17:52:31ZBiophysical and structural studies reveal marginal stability of a crucial hydrocarbon biosynthetic enzyme acyl ACP reductase10.1038/s41598-021-91232-02045-2322https://doaj.org/article/f138d43b90734f25a7dca822c071ff852021-06-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-91232-0https://doaj.org/toc/2045-2322Abstract Acyl-ACP reductase (AAR) is one of the two key cyanobacterial enzymes along with aldehyde deformylating oxygenase (ADO) involved in the synthesis of long-chain alkanes, a drop-in biofuel. The enzyme is prone to aggregation when expressed in Escherichia coli, leading to varying alkane levels. The present work attempts to investigate the crucial structural aspects of AAR protein associated with its stability and folding. Characterization by dynamic light scattering experiment and intact mass spectrometry revealed that recombinantly expressed AAR in E. coli existed in multiple-sized protein particles due to diverse lipidation. Interestingly, while thermal- and urea-based denaturation of AAR showed 2-state unfolding transition in circular dichroism and intrinsic fluorescent spectroscopy, the unfolding process of AAR was a 3-state pathway in GdnHCl solution suggesting that the protein milieu plays a significant role in dictating its folding. Apparent standard free energy $$\left( {\Delta {\text{G}}_{{{\text{NU}}}}^{{{\text{H}}_{2} {\text{O}}}} } \right)$$ Δ G NU H 2 O of ~ 4.5 kcal/mol for the steady-state unfolding of AAR indicated borderline stability of the protein. Based on these evidences, we propose that the marginal stability of AAR are plausible contributing reasons for aggregation propensity and hence the low catalytic activity of the enzyme when expressed in E. coli for biofuel production. Our results show a path for building superior biocatalyst for higher biofuel production.Ashima SharmaTabinda ShakeelMayank GuptaGirish H. RajacharyaSyed Shams YazdaniNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-21 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Ashima Sharma Tabinda Shakeel Mayank Gupta Girish H. Rajacharya Syed Shams Yazdani Biophysical and structural studies reveal marginal stability of a crucial hydrocarbon biosynthetic enzyme acyl ACP reductase
description	Abstract Acyl-ACP reductase (AAR) is one of the two key cyanobacterial enzymes along with aldehyde deformylating oxygenase (ADO) involved in the synthesis of long-chain alkanes, a drop-in biofuel. The enzyme is prone to aggregation when expressed in Escherichia coli, leading to varying alkane levels. The present work attempts to investigate the crucial structural aspects of AAR protein associated with its stability and folding. Characterization by dynamic light scattering experiment and intact mass spectrometry revealed that recombinantly expressed AAR in E. coli existed in multiple-sized protein particles due to diverse lipidation. Interestingly, while thermal- and urea-based denaturation of AAR showed 2-state unfolding transition in circular dichroism and intrinsic fluorescent spectroscopy, the unfolding process of AAR was a 3-state pathway in GdnHCl solution suggesting that the protein milieu plays a significant role in dictating its folding. Apparent standard free energy $$\left( {\Delta {\text{G}}_{{{\text{NU}}}}^{{{\text{H}}_{2} {\text{O}}}} } \right)$$ Δ G NU H 2 O of ~ 4.5 kcal/mol for the steady-state unfolding of AAR indicated borderline stability of the protein. Based on these evidences, we propose that the marginal stability of AAR are plausible contributing reasons for aggregation propensity and hence the low catalytic activity of the enzyme when expressed in E. coli for biofuel production. Our results show a path for building superior biocatalyst for higher biofuel production.
format	article
author	Ashima Sharma Tabinda Shakeel Mayank Gupta Girish H. Rajacharya Syed Shams Yazdani
author_facet	Ashima Sharma Tabinda Shakeel Mayank Gupta Girish H. Rajacharya Syed Shams Yazdani
author_sort	Ashima Sharma
title	Biophysical and structural studies reveal marginal stability of a crucial hydrocarbon biosynthetic enzyme acyl ACP reductase
title_short	Biophysical and structural studies reveal marginal stability of a crucial hydrocarbon biosynthetic enzyme acyl ACP reductase
title_full	Biophysical and structural studies reveal marginal stability of a crucial hydrocarbon biosynthetic enzyme acyl ACP reductase
title_fullStr	Biophysical and structural studies reveal marginal stability of a crucial hydrocarbon biosynthetic enzyme acyl ACP reductase
title_full_unstemmed	Biophysical and structural studies reveal marginal stability of a crucial hydrocarbon biosynthetic enzyme acyl ACP reductase
title_sort	biophysical and structural studies reveal marginal stability of a crucial hydrocarbon biosynthetic enzyme acyl acp reductase
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/f138d43b90734f25a7dca822c071ff85
work_keys_str_mv	AT ashimasharma biophysicalandstructuralstudiesrevealmarginalstabilityofacrucialhydrocarbonbiosyntheticenzymeacylacpreductase AT tabindashakeel biophysicalandstructuralstudiesrevealmarginalstabilityofacrucialhydrocarbonbiosyntheticenzymeacylacpreductase AT mayankgupta biophysicalandstructuralstudiesrevealmarginalstabilityofacrucialhydrocarbonbiosyntheticenzymeacylacpreductase AT girishhrajacharya biophysicalandstructuralstudiesrevealmarginalstabilityofacrucialhydrocarbonbiosyntheticenzymeacylacpreductase AT syedshamsyazdani biophysicalandstructuralstudiesrevealmarginalstabilityofacrucialhydrocarbonbiosyntheticenzymeacylacpreductase
_version_	1718379204817453056

Biophysical and structural studies reveal marginal stability of a crucial hydrocarbon biosynthetic enzyme acyl ACP reductase

Ejemplares similares