Purification and characterization of two polymorphic variants of short chain acyl-CoA dehydrogenase reveal reduction of catalytic activity and stability of the Gly185Ser enzyme

Tien V. Nguyen, Charles Riggs, Dusica Babovic-Vuksanovic, Yong Sung Kim, John F. Carpenter, Thomas P Burghardt, Niels Gregersen, Jerry Vockley

Research output: Contribution to journalArticle

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Abstract

Short chain acyl-CoA dehydrogenase (SCAD) is a homotetrameric flavoenzyme that catalyzes the first intramitochondrial step in the β-oxidation of fatty acids. Two polymorphisms in the coding region of the SCAD gene, 511C>T (R147W) and 625G>A (G185S), have been shown to be associated with an increased level of ethylmalonic acid excretion in urine, a clinical characteristic of SCAD deficiency. To characterize the biochemical consequences of these variations, in vitro site-directed mutagenesis and prokaryotic expression were used to produce the corresponding SCAD variant proteins. Both variant proteins were unstable when produced in Escherichia coli, but could be rescued and subsequently purified by coexpressing them with the bacterial chaperonin GroEL/ES. The kcat/Km values of the green wild-type, R147W, and G185S SCAD enzymes coexpressed with GroEL/ES were 33, 30, and 10 μM-1 s-1, respectively. There were minimal differences in the kinetic parameters measured for the green, degreened, and wild-type enzymes coexpressed with GroEL/ES, and the R147W variant when butyryl-CoA was used as a substrate. The catalytic efficiency of the G185S variant enzyme, however, was reduced compared to that of the wild-type enzyme. The thermal and guanidine HCl stability of the purified enzymes as determined by fluorescence, far-UV CD spectroscopy, and incubation-induced rest activity showed the following order of relative stability: wild-type enzyme > R147W > G185S. Near-UV CD spectroscopy indicated that these impairments are caused by decreased flexibility in the tertiary conformation of the two mutant enzymes. The common SCAD polymorphisms may lead to clinically relevant alterations in enzyme function.

Original languageEnglish (US)
Pages (from-to)11126-11133
Number of pages8
JournalBiochemistry
Volume41
Issue number37
DOIs
StatePublished - Sep 17 2002

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Butyryl-CoA Dehydrogenase
Enzyme Stability
Purification
Catalyst activity
Enzymes
Spectrum Analysis
Polymorphism
Chaperonins
Guanidine
Spectroscopy
Site-Directed Mutagenesis
Mutagenesis
Proteins
Fatty Acids
Hot Temperature
Fluorescence
Kinetic parameters
Urine
Escherichia coli
Conformations

ASJC Scopus subject areas

  • Biochemistry

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Purification and characterization of two polymorphic variants of short chain acyl-CoA dehydrogenase reveal reduction of catalytic activity and stability of the Gly185Ser enzyme. / Nguyen, Tien V.; Riggs, Charles; Babovic-Vuksanovic, Dusica; Kim, Yong Sung; Carpenter, John F.; Burghardt, Thomas P; Gregersen, Niels; Vockley, Jerry.

In: Biochemistry, Vol. 41, No. 37, 17.09.2002, p. 11126-11133.

Research output: Contribution to journalArticle

Nguyen, Tien V. ; Riggs, Charles ; Babovic-Vuksanovic, Dusica ; Kim, Yong Sung ; Carpenter, John F. ; Burghardt, Thomas P ; Gregersen, Niels ; Vockley, Jerry. / Purification and characterization of two polymorphic variants of short chain acyl-CoA dehydrogenase reveal reduction of catalytic activity and stability of the Gly185Ser enzyme. In: Biochemistry. 2002 ; Vol. 41, No. 37. pp. 11126-11133.
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abstract = "Short chain acyl-CoA dehydrogenase (SCAD) is a homotetrameric flavoenzyme that catalyzes the first intramitochondrial step in the β-oxidation of fatty acids. Two polymorphisms in the coding region of the SCAD gene, 511C>T (R147W) and 625G>A (G185S), have been shown to be associated with an increased level of ethylmalonic acid excretion in urine, a clinical characteristic of SCAD deficiency. To characterize the biochemical consequences of these variations, in vitro site-directed mutagenesis and prokaryotic expression were used to produce the corresponding SCAD variant proteins. Both variant proteins were unstable when produced in Escherichia coli, but could be rescued and subsequently purified by coexpressing them with the bacterial chaperonin GroEL/ES. The kcat/Km values of the green wild-type, R147W, and G185S SCAD enzymes coexpressed with GroEL/ES were 33, 30, and 10 μM-1 s-1, respectively. There were minimal differences in the kinetic parameters measured for the green, degreened, and wild-type enzymes coexpressed with GroEL/ES, and the R147W variant when butyryl-CoA was used as a substrate. The catalytic efficiency of the G185S variant enzyme, however, was reduced compared to that of the wild-type enzyme. The thermal and guanidine HCl stability of the purified enzymes as determined by fluorescence, far-UV CD spectroscopy, and incubation-induced rest activity showed the following order of relative stability: wild-type enzyme > R147W > G185S. Near-UV CD spectroscopy indicated that these impairments are caused by decreased flexibility in the tertiary conformation of the two mutant enzymes. The common SCAD polymorphisms may lead to clinically relevant alterations in enzyme function.",
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T1 - Purification and characterization of two polymorphic variants of short chain acyl-CoA dehydrogenase reveal reduction of catalytic activity and stability of the Gly185Ser enzyme

AU - Nguyen, Tien V.

AU - Riggs, Charles

AU - Babovic-Vuksanovic, Dusica

AU - Kim, Yong Sung

AU - Carpenter, John F.

AU - Burghardt, Thomas P

AU - Gregersen, Niels

AU - Vockley, Jerry

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N2 - Short chain acyl-CoA dehydrogenase (SCAD) is a homotetrameric flavoenzyme that catalyzes the first intramitochondrial step in the β-oxidation of fatty acids. Two polymorphisms in the coding region of the SCAD gene, 511C>T (R147W) and 625G>A (G185S), have been shown to be associated with an increased level of ethylmalonic acid excretion in urine, a clinical characteristic of SCAD deficiency. To characterize the biochemical consequences of these variations, in vitro site-directed mutagenesis and prokaryotic expression were used to produce the corresponding SCAD variant proteins. Both variant proteins were unstable when produced in Escherichia coli, but could be rescued and subsequently purified by coexpressing them with the bacterial chaperonin GroEL/ES. The kcat/Km values of the green wild-type, R147W, and G185S SCAD enzymes coexpressed with GroEL/ES were 33, 30, and 10 μM-1 s-1, respectively. There were minimal differences in the kinetic parameters measured for the green, degreened, and wild-type enzymes coexpressed with GroEL/ES, and the R147W variant when butyryl-CoA was used as a substrate. The catalytic efficiency of the G185S variant enzyme, however, was reduced compared to that of the wild-type enzyme. The thermal and guanidine HCl stability of the purified enzymes as determined by fluorescence, far-UV CD spectroscopy, and incubation-induced rest activity showed the following order of relative stability: wild-type enzyme > R147W > G185S. Near-UV CD spectroscopy indicated that these impairments are caused by decreased flexibility in the tertiary conformation of the two mutant enzymes. The common SCAD polymorphisms may lead to clinically relevant alterations in enzyme function.

AB - Short chain acyl-CoA dehydrogenase (SCAD) is a homotetrameric flavoenzyme that catalyzes the first intramitochondrial step in the β-oxidation of fatty acids. Two polymorphisms in the coding region of the SCAD gene, 511C>T (R147W) and 625G>A (G185S), have been shown to be associated with an increased level of ethylmalonic acid excretion in urine, a clinical characteristic of SCAD deficiency. To characterize the biochemical consequences of these variations, in vitro site-directed mutagenesis and prokaryotic expression were used to produce the corresponding SCAD variant proteins. Both variant proteins were unstable when produced in Escherichia coli, but could be rescued and subsequently purified by coexpressing them with the bacterial chaperonin GroEL/ES. The kcat/Km values of the green wild-type, R147W, and G185S SCAD enzymes coexpressed with GroEL/ES were 33, 30, and 10 μM-1 s-1, respectively. There were minimal differences in the kinetic parameters measured for the green, degreened, and wild-type enzymes coexpressed with GroEL/ES, and the R147W variant when butyryl-CoA was used as a substrate. The catalytic efficiency of the G185S variant enzyme, however, was reduced compared to that of the wild-type enzyme. The thermal and guanidine HCl stability of the purified enzymes as determined by fluorescence, far-UV CD spectroscopy, and incubation-induced rest activity showed the following order of relative stability: wild-type enzyme > R147W > G185S. Near-UV CD spectroscopy indicated that these impairments are caused by decreased flexibility in the tertiary conformation of the two mutant enzymes. The common SCAD polymorphisms may lead to clinically relevant alterations in enzyme function.

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