Analysis of Enoyl-Coenzyme A Hydratase Activity and Its Stereospecificity Using High-Performance Liquid Chromatography Equipped with Chiral Separation Column

Tsuchida, Shirou; Kawamoto, Koutarou; Nunome, Kana; Hamaue, Naoya; Yoshimura, Teizo; Aoki, Takashi; Kurosawa, Takao

doi:10.5650/jos.60.221

Cited by 7 publications

(11 citation statements)

References 28 publications

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“…The substrate ( trans -2-hexadecenoyl-CoA) and the product (3 R -hydroxyhexadecanoyl-CoA) were synthesized chemically as described previously [6]. All other materials and chemicals were of analytical grade.…”

Section: Methodsmentioning

confidence: 99%

“…To aid in the investigation of the physiological significance of the stereospecificity of d - and l -bifunctional protein ( l -BP), we have developed a system for separate quantification of 3 R - and 3 S -hydroxyacyl-CoA, which is a product of l -BP hydratase, using high-performance liquid chromatography (HPLC) with a chiral separation column [6]. This method enables us to analyze the activity of hydratase even if d -BP and l -BP are both present in the experimental system.…”

Section: Introductionmentioning

confidence: 99%

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Effects of naturally occurring missense mutations and G525V in the hydratase domain of human d-bifunctional protein on hydratase activity

Tsuchida

Osaka

Abe

et al. 2015

Molecular Genetics and Metabolism Reports

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d-bifunctional protein (d-BP) deficiency is thought to lead to severe lipid metabolism disorders. To investigate the effect of naturally occurring missense mutations in the hydratase domain in d-BP, we constructed several d-BP hydratase variants and measured their activities. Missense mutations at sites whose conservation rates among 30 eukaryotes were < 70% did not affect hydratase activity. We predicted that missense mutations of highly conserved amino acids would markedly reduce activity. However, R562H and R562L, naturally occurring missense mutations of highly conserved amino acids, did not reduce activity. This result suggests that a missense mutation in a highly conserved amino acid does not always lead to severe lipid metabolism disorders. We also investigated the effect of G525V, which had been found in a mildly symptomatic patient with d-BP deficiency who was heterozygous for G525 and G658X. G525V markedly reduced hydratase activity. We had predicted that heterozygous G525V and G658X would lead to severely disordered lipid metabolism. However, the symptoms were inconsistent with this prediction. Characterizing mutations in the d-BP gene and the symptoms of d-BP deficiency may require pleiotropy, not only in vitro, studies.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Effects of naturally occurring missense mutations and G525V in the hydratase domain of human d-bifunctional protein on hydratase activity

Tsuchida

Osaka

Abe

et al. 2015

Molecular Genetics and Metabolism Reports

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“…The R -3-hydroxyhexadecanoyl-CoA 8 , S -3-hydroxyhexadecanoyl-CoA 9 , and trans-2-hexadecenoylCoA 10 were separated under HPLC conditions that have been described in previous article 9 . Additionally, the 3-oxohexadecanoyl-CoA 7 was mixed with other CoA esters and subjected to HPLC under the reported conditions.…”

Section: Separation Of the Coa Esters On Hplcmentioning

confidence: 99%

“…Ltd., Tokyo, Japan was used as the separation column. R -3-hydroxyhexadecanoyl-CoA, S -3-hydroxyhexadecanoyl-CoA and trans-2-hexadecenoyl-CoA were prepared using our procedures described in previous reports 8,9 .…”

Section: Materials and Instrumentsmentioning

confidence: 99%

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Synthesis and Detection by HPLC of 3-Oxohexadecanoyl-CoA for the Study of Peroxisomal Bifunctional Proteins

et al. 2017

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A chiral high-performance liquid chromatography–tandem mass spectrometry method for the stereospecific analysis of enoyl-coenzyme A hydratases/isomerases

Abdel-Mawgoud¹,

Lépine²,

Déziel³

2013

Journal of Chromatography A

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Analysis of Enoyl-Coenzyme A Hydratase Activity and Its Stereospecificity Using High-Performance Liquid Chromatography Equipped with Chiral Separation Column

Cited by 7 publications

References 28 publications

Effects of naturally occurring missense mutations and G525V in the hydratase domain of human d-bifunctional protein on hydratase activity

Effects of naturally occurring missense mutations and G525V in the hydratase domain of human d-bifunctional protein on hydratase activity

Synthesis and Detection by HPLC of 3-Oxohexadecanoyl-CoA for the Study of Peroxisomal Bifunctional Proteins

A chiral high-performance liquid chromatography–tandem mass spectrometry method for the stereospecific analysis of enoyl-coenzyme A hydratases/isomerases

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