Functional assignment of Glu386 and Arg388 in the active site of l-galactono-γ-lactone dehydrogenase

Nicole G.H. Leferink; Mac Donald F. Jose; Willy A.M. van den Berg; Willem J.H. van Berkel

doi:10.1016/j.febslet.2009.09.004

Functional assignment of Glu386 and Arg388 in the active site of l-galactono-γ-lactone dehydrogenase

Nicole G.H. Leferink, Mac Donald F. Jose, Willy A.M. van den Berg, Willem J.H. van Berkel^*

^*Corresponding author for this work

Wageningen University

Research output: Contribution to journal › Article › peer-review

Abstract

The flavoenzyme l-galactono-γ-lactone dehydrogenase (GALDH) catalyzes the terminal step of vitamin C biosynthesis in plants. Little is known about the catalytic mechanism of GALDH and related aldonolactone oxidoreductases. Here we identified an essential Glu-Arg pair in the active site of GALDH from Arabidopsis thaliana. Glu386 and Arg388 variants show high K_m values for l-galactono-1,4-lactone and low turnover rates. Arg388 is crucial for the stabilization of the anionic form of the reduced FAD cofactor. Glu386 is involved in productive substrate binding. The E386D variant has lost its specificity for l-galactono-1,4-lactone and shows the highest catalytic efficiency with l-gulono-1,4-lactone.

Original language	English
Pages (from-to)	3199-3203
Number of pages	5
Journal	FEBS Letters
Volume	583
Issue number	19
DOIs	https://doi.org/10.1016/j.febslet.2009.09.004
Publication status	Published - 6 Oct 2009

Keywords

Flavoprotein
l-Galactono-1,4-lactone dehydrogenase (EC 1.3.2.3)
Site-directed mutagenesis
VAO family
Vitamin C

Access to Document

10.1016/j.febslet.2009.09.004

Cite this

@article{84d5a35b3c9d460ebd89e748a2364539,

title = "Functional assignment of Glu386 and Arg388 in the active site of l-galactono-γ-lactone dehydrogenase",

abstract = "The flavoenzyme l-galactono-γ-lactone dehydrogenase (GALDH) catalyzes the terminal step of vitamin C biosynthesis in plants. Little is known about the catalytic mechanism of GALDH and related aldonolactone oxidoreductases. Here we identified an essential Glu-Arg pair in the active site of GALDH from Arabidopsis thaliana. Glu386 and Arg388 variants show high Km values for l-galactono-1,4-lactone and low turnover rates. Arg388 is crucial for the stabilization of the anionic form of the reduced FAD cofactor. Glu386 is involved in productive substrate binding. The E386D variant has lost its specificity for l-galactono-1,4-lactone and shows the highest catalytic efficiency with l-gulono-1,4-lactone.",

keywords = "Flavoprotein, l-Galactono-1,4-lactone dehydrogenase (EC 1.3.2.3), Site-directed mutagenesis, VAO family, Vitamin C",

author = "Leferink, {Nicole G.H.} and Jose, {Mac Donald F.} and {van den Berg}, {Willy A.M.} and {van Berkel}, {Willem J.H.}",

year = "2009",

month = oct,

day = "6",

doi = "10.1016/j.febslet.2009.09.004",

language = "English",

volume = "583",

pages = "3199--3203",

journal = "FEBS Letters",

issn = "0014-5793",

publisher = "John Wiley & Sons Ltd",

number = "19",

}

TY - JOUR

T1 - Functional assignment of Glu386 and Arg388 in the active site of l-galactono-γ-lactone dehydrogenase

AU - Leferink, Nicole G.H.

AU - Jose, Mac Donald F.

AU - van den Berg, Willy A.M.

AU - van Berkel, Willem J.H.

PY - 2009/10/6

Y1 - 2009/10/6

N2 - The flavoenzyme l-galactono-γ-lactone dehydrogenase (GALDH) catalyzes the terminal step of vitamin C biosynthesis in plants. Little is known about the catalytic mechanism of GALDH and related aldonolactone oxidoreductases. Here we identified an essential Glu-Arg pair in the active site of GALDH from Arabidopsis thaliana. Glu386 and Arg388 variants show high Km values for l-galactono-1,4-lactone and low turnover rates. Arg388 is crucial for the stabilization of the anionic form of the reduced FAD cofactor. Glu386 is involved in productive substrate binding. The E386D variant has lost its specificity for l-galactono-1,4-lactone and shows the highest catalytic efficiency with l-gulono-1,4-lactone.

AB - The flavoenzyme l-galactono-γ-lactone dehydrogenase (GALDH) catalyzes the terminal step of vitamin C biosynthesis in plants. Little is known about the catalytic mechanism of GALDH and related aldonolactone oxidoreductases. Here we identified an essential Glu-Arg pair in the active site of GALDH from Arabidopsis thaliana. Glu386 and Arg388 variants show high Km values for l-galactono-1,4-lactone and low turnover rates. Arg388 is crucial for the stabilization of the anionic form of the reduced FAD cofactor. Glu386 is involved in productive substrate binding. The E386D variant has lost its specificity for l-galactono-1,4-lactone and shows the highest catalytic efficiency with l-gulono-1,4-lactone.

KW - Flavoprotein

KW - l-Galactono-1,4-lactone dehydrogenase (EC 1.3.2.3)

KW - Site-directed mutagenesis

KW - VAO family

KW - Vitamin C

UR - http://www.scopus.com/inward/record.url?scp=70349464664&partnerID=8YFLogxK

U2 - 10.1016/j.febslet.2009.09.004

DO - 10.1016/j.febslet.2009.09.004

M3 - Article

C2 - 19737562

AN - SCOPUS:70349464664

SN - 0014-5793

VL - 583

SP - 3199

EP - 3203

JO - FEBS Letters

JF - FEBS Letters

IS - 19

ER -

Functional assignment of Glu386 and Arg388 in the active site of l-galactono-γ-lactone dehydrogenase

Abstract

Keywords

Access to Document

Other files and links

Fingerprint

Cite this