Preparation and characterization of EGCase I, applicable to the comprehensive analysis of GSLs, using a rhodococcal expression system

Yohei Ishibashi, Utaro Kobayashi, Atsushi Hijikata, Keishi Sakaguchi, Hatsumi M. Goda, Tomohiro Tamura, Nozomu Okino, Makoto Ito

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Abstract

Endoglycoceramidase (EGCase) is a glycosidase capable of hydrolyzing the β-glycosidic linkage between the oligosaccharides and ceramides of glycosphingolipids (GSLs). Three molecular species of EGCase differing in specificity were found in the culture fluid of Rhodococcus equi (formerly Rhodococcus sp. M-750) and designated EGCase I, II, and III. This study describes the molecular cloning of EGCase I and characterization of the recombinant enzyme, which was highly expressed in a rhodococcal expression system using Rhodococcus erythropolis. Kinetic analysis revealed the turnover number (kcat) (kcat) of the recombinant EGCase I to be 22-and 1,200-fold higher than that of EGCase II toward GM1a and Gb3Cer, respectively, although the Km of both enzymes was almost the same for these substrates. Comparison of the three-dimensional structure of EGCase I (model) and EGCase II (crystal) indicated that a flexible loop hangs over the catalytic cleft of EGCase II but not EGCase I. Deletion of the loop from EGCase II increased the kcat of the mutant enzyme, suggesting that the loop is a critical factor affecting the turnover of substrates and products in the catalytic region. Recombinant EGCase I exhibited broad specificity and good reaction efficiency compared with EGCase II, making EGCase I well-suited to a comprehensive analysis of GSLs.

Original languageEnglish
Pages (from-to)2242-2251
Number of pages10
JournalJournal of Lipid Research
Volume53
Issue number10
DOIs
Publication statusPublished - Oct 1 2012

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endoglycoceramidase
Glycosphingolipids
Rhodococcus
Enzymes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Endocrinology
  • Cell Biology

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Preparation and characterization of EGCase I, applicable to the comprehensive analysis of GSLs, using a rhodococcal expression system. / Ishibashi, Yohei; Kobayashi, Utaro; Hijikata, Atsushi; Sakaguchi, Keishi; Goda, Hatsumi M.; Tamura, Tomohiro; Okino, Nozomu; Ito, Makoto.

In: Journal of Lipid Research, Vol. 53, No. 10, 01.10.2012, p. 2242-2251.

Research output: Contribution to journalArticle

Ishibashi, Yohei ; Kobayashi, Utaro ; Hijikata, Atsushi ; Sakaguchi, Keishi ; Goda, Hatsumi M. ; Tamura, Tomohiro ; Okino, Nozomu ; Ito, Makoto. / Preparation and characterization of EGCase I, applicable to the comprehensive analysis of GSLs, using a rhodococcal expression system. In: Journal of Lipid Research. 2012 ; Vol. 53, No. 10. pp. 2242-2251.
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