The pathway via D-galacturonate/L-galactonate is significant for ascorbate biosynthesis in Euglena gracilis: identification and functional characterization of aldonolactonase.
We have previously proposed that Euglena gracilis possesses a pathway for the production of ascorbate (AsA) through d-galacturonate/L-galactonate as representative intermediates ( Shigeoka, S., Nakano, Y., and Kitaoka, S. (1979) J. Nutr. Sci. Vitaminol. 25, 299-307 ). However, genetic evidence proving that the pathway exists has not been obtained yet. We report here the identification of a gene encoding aldonolactonase, which catalyzes a penultimate step of the biosynthesis of AsA in Euglena. By a BLAST search, we identified one candidate for the enzyme having significant sequence identity with rat gluconolactonase, a key enzyme for the production of AsA via d-glucuronate in animals. The purified recombinant aldonolactonase expressed in Escherichia coli catalyzed the reversible reaction of L-galactonate and L-galactono-1,4-lactone with zinc ion as a cofactor. The apparent K(m) values for L-galactonate and L-galactono-1,4-lactone were 1.55 +/- 0.3 and 1.67 +/- 0.39 mm, respectively. The cell growth of Euglena was arrested by silencing the expression of aldonolactonase through RNA interference and then restored to the normal state by supplementation with L-galactono-1,4-lactone. Euglena cells accumulated more AsA on supplementation with d-galacturonate than d-glucuronate. The present results indicate that aldonolactonase is significant for the biosynthesis of AsA in Euglena cells, which predominantly utilize the pathwayviad-galacturonate/L-galactonate. The identification of aldonolactonase provides the first insight into the biosynthesis of AsA via uronic acids as the intermediate in photosynthetic algae including Euglena.
Ishikawa
Takahiro
T
Faculty of Life and Environmental Science, Shimane University, Matsue, Shimane 690-8504, Japan. ishikawa@life.shimane-u.ac.jp
Nishikawa
Hitoshi
H
Gao
Youngshun
Y
Sawa
Yoshihiro
Y
Shibata
Hitoshi
H
Yabuta
Yukinori
Y
Maruta
Takanori
T
Shigeoka
Shigeru
S
D020418:Algal Proteins / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001205:Ascorbic Acid / Q000096:biosynthesis* / Q000235:genetics; D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005056:Euglena gracilis / Q000201:enzymology* / Q000235:genetics; D006603:Hexuronic Acids / Q000378:metabolism*; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013400:Sugar Acids / Q000378:metabolism*
D020418:Algal Proteins; D006603:Hexuronic Acids; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D013400:Sugar Acids; C012991:galactonic acid; C007819:galacturonic acid; D002265:Carboxylic Ester Hydrolases; C020579:gluconolactonase; D001205:Ascorbic Acid
Plant J. 2007 Nov;52(4):673-89
17877701
Plant Physiol. 2000 May;123(1):335-44
10806250
Anal Biochem. 1995 Feb 10;225(1):165-7
7778771
Proc Natl Acad Sci U S A. 2006 Apr 11;103(15):5723-8
16585534
Annu Rev Plant Physiol Plant Mol Biol. 2001 Jun;52:437-467
11337405
J Biol Chem. 2007 Feb 23;282(8):5770-80
17166853
J Exp Bot. 2003 Aug;54(389):1841-9
12869519
Biosci Biotechnol Biochem. 2008 May;72(5):1143-54
18460785
Proc Natl Acad Sci U S A. 2005 Dec 6;102(49):17606-11
16314577
Plant Physiol. 2004 Mar;134(3):1200-5
14976233
Eur J Biochem. 1992 Oct 1;209(1):383-90
1396712
Biosci Biotechnol Biochem. 2006 Nov;70(11):2720-6
17090924
Proc Natl Acad Sci U S A. 2001 Feb 27;98(5):2262-7
11226227
J Protozool. 1967 May;14(2):238-44
6038033
J Nutr Sci Vitaminol (Tokyo). 1979;25(4):299-307
118242
J Biol Chem. 2006 Jun 9;281(23):15662-70
16595667
Nat Biotechnol. 2003 Feb;21(2):177-81
12524550
Biochem Pharmacol. 1993 May 25;45(10):2087-91
8390260
Biochim Biophys Acta. 1972 Jul 13;276(1):176-9
4625870
J Exp Bot. 2002 May;53(372):1305-19
11997377
Plant Cell Physiol. 2000 Jun;41(6):666-75
10945335
Subcell Biochem. 1996;25:17-39
8821967
Plant Cell Physiol. 2004 Sep;45(9):1271-9
15509850
Proc Natl Acad Sci U S A. 1999 Mar 30;96(7):4198-203
10097187
Nature. 2002 Feb 28;415(6875):1047-51
11875575
Curr Opin Plant Biol. 2000 Jun;3(3):229-35
10837263
J Cell Sci. 1993 Apr;104 ( Pt 4):1163-73
8391014
J Photochem Photobiol B. 2006 Sep 1;84(3):197-203
16679025
Proc Natl Acad Sci U S A. 2005 Aug 16;102(33):11645-50
16087875
Biochim Biophys Acta. 1996 May 21;1290(1):69-75
8645709
J Ind Microbiol Biotechnol. 2002 Aug;29(2):93-8
12161776
J Cyclic Nucleotide Res. 1979;5(2):107-23
36416
J Biol Chem. 2003 Nov 28;278(48):47483-90
12954627