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Identification of the Catalytic Residue of Rat Acyl-CoA Dehydrogenase 9 by Site-Directed Mutagenesis

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Abstract

Acyl-CoA dehydrogenase 9 (ACAD 9) is the ninth member of ACADs involved in mitochondrial fatty acid oxidation and possibly complex I assembly. Sequence alignment suggested that Glu389 of rat ACAD 9 was highly conserved and located near the active center and might act as an important base for the dehydrogenation reaction. The role of Glu389 in the catalytic reaction was investigated by site-directed mutagenesis. Both wild-type and mutant ACAD 9 proteins were purified and their catalytic characterization was studied. When Glu389 was replaced by other residues, the enzyme activity could be lost to a large extent. Those results suggested that Glu389 could function as the catalytic base that abstracted the α-proton of the acyl-CoA substrate in a proposed catalytic mechanism.

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Abbreviations

ACAD 9:

Acyl-CoA Dehydrogenase 9

PAGE:

Polyacrylamide gel electrophoresis

PCR:

Polymerase chain reaction

SDS:

Sodium dodecyl sulfate

ETF:

Electron transfer flavoprotein

E. coli :

Escherichia coli

OD600 :

Optical density at 600 nm

IPTG:

Isopropyl-D-thiogalactopyranoside

PMS:

Phenazine methosulfate

DCPIP:

2,6-dichlorophenolindophenol

MS:

Mass spectrometry

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Acknowledgements

This work was supported by the National Natural Science Fund of P. R. China (30900024), the Doctoral Program of Ministry of Education of China (200805331034), and the Doctoral Scientific Research Foundation of Hunan University of Science and Technology (E51691).

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Correspondence to Jia Zeng.

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Zeng, J., Deng, S. & Wang, Y. Identification of the Catalytic Residue of Rat Acyl-CoA Dehydrogenase 9 by Site-Directed Mutagenesis. Appl Biochem Biotechnol 182, 1198–1207 (2017). https://doi.org/10.1007/s12010-016-2392-1

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