Tài liệu Báo cáo khóa học: Characterization of the products of the genes SNO1 and SNZ1 involved in

Characterization of the products of the genes SNO1 and SNZ1 involved

in pyridoxine synthesis in Saccharomyces cerevisiae

Yi-Xin Dong, Shinji Sueda, Jun-Ichi Nikawa and Hiroki Kondo

Department of Biochemical Engineering and Science, Kyushu Institute of Technology, Iizuka, Japan

Genes SNO1 and SNZ1 are Saccharomyces cerevisiae

homologues of PDX2 and PDX1 which participate in pyri￾doxine synthesis in the fungus Cercospora nicotianae. In

order to clarify their function, the two genes SNO1 and

SNZ1 were expressed in Escherichia coli either individually

or simultaneously and with or without a His-tag. When

expressed simultaneously, the two protein products formed

a complex and showed glutaminase activity. When purified

to homogeneity, the complex exhibited a specific activity of

480 nmolÆmg)1

Æmin)1 as glutaminase, with a Km of 3.4 mM

for glutamine. These values are comparable to those for

other glutamine amidotransferases. In addition, the gluta￾minase activity was impaired by 6-diazo-5-oxo-L-norleucine

in a time- and dose-dependent manner and the enzyme was

protected from deactivation by glutamine. These data sug￾gest strongly that the complex of Sno1p and Snz1p is a

glutamine amidotransferase with the former serving as the

glutaminase, although the activity was barely detectable with

Sno1p alone. The function of Snz1p and the amido acceptor

for ammonia remain to be identified.

Keywords: glutamine amidotransferase; pyridoxine biosyn￾thesis; Saccharomyces cerevisiae; SNO1; SNZ1.

Pyridoxal phosphate plays a crucial role in amino acid

metabolism. Pyridoxine and its phosphate are the precur￾sors of pyridoxal phosphate and the major forms of vitamin

B6. Pyridoxine biosynthesis in Escherichia coli has been

studied extensively but only recently has the whole synthetic

pathway been finally established [1–3]. There are organisms

such as budding yeast, Saccharomyces cerevisiae, which

also synthesize pyridoxine but in a different pathway. This

notion is based in part on an observation that the nitrogen

of pyridoxine is derived from the amide group of glutamine

in yeast [4], while glutamate is the source of the ring nitrogen

in E. coli [5]. Recently, two independent groups identified

pyroA and SOR1 (PDX1) as participating in pyridoxine

synthesis in fungi Cercospora nicotianae and Aspergillus

nidulans, respectively [6,7]. They are homologous genes and

their homologues are distributed widely in various organ￾isms, but nothing of their function is known except that

SNZ1, the yeast homologue, works in the stationary phase

of yeast cells together with SNO1 [8]. In addition to these

observations, it was shown recently that a pentose or

pentulose constitutes the skeleton of pyridoxine in yeast

[9,10]. Herein, we report that Sno1p and Snz1p serve as a

glutaminase to supply ammonia for the ring nitrogen of

pyridoxine in yeast. Based on these and other lines of

evidence, a putative synthetic pathway to pyridoxine is

presented in the Discussion in which ribulose 5-phosphate

and ammonia serve as the key starting or intermediary

material.

Experimental procedures

Materials

Inorganic salts and common organic chemicals including

amino acids, nucleic bases and vitamins were obtained from

commercial sources. Acetylpyridine adenine dinucleotide

(APAD) and 6-diazo-5-oxo-L-norleucine (DON) were from

Sigma (St. Louis, MO, USA). Glutamate dehydrogenase

from bovine liver was obtained from Oriental Yeast (Tokyo,

Japan). Reagents for genetic engineering such as restriction

enzymes were purchased from Takara (Kyoto, Japan) and

New England Biolabs (Beverly, MA, USA). Oligonucleo￾tides were custom synthesized by Hokkaido Science (Sap￾poro, Japan). Plasmid YEpM4 was a 2 lm DNA-based

shuttle vector with gene LEU2 as the selectable marker [11].

Plasmids pET21a, pET21d (both ampicillin resistant),

pET24a (kanamycin resistant) and His-bind columns were

from Novagen (Madison, WI, USA). The TOPO TA

cloning kit was the product of Invitrogen (Carlsbad, CA,

USA).

Strains and media

The S. cerevisiae strain used in this study was D373-1

(MATa, leu2, his3, trp1) [12]. The following medium was

used to grow yeast: YPD [1% yeast extract, 2% polypep￾tone, 2% glucose (v/v/v)] and synthetic medium [glucose,

20 g; (NH4)2SO4, 1.02 g; KH2PO4, 0.875 g; K2HPO4,

0.125 g; CaCl2ÆH2O, 0.02 g; NaCl, 0.01 g; MgSO4Æ7H2O,

0.05 g; CuSO4Æ5H2O, 40 lg; MnSO4ÆH2O, 400 lg; FeCl3Æ

6H2O, 200 lg; ZnSO4Æ7H2O, 400 lg; Na2MoO4Æ2H2O,

Correspondence to H. Kondo, Department of Biochemical

Engineering and Science, Kyushu Institute of Technology,

Kawazu 680-4, Iizuka 820-8502, Japan.

Fax: + 81 948 7801, Tel.: + 81 948 29 7814,

E-mail: [email protected]

Abbreviations: APAD, acetylpyridine adenine dinucleotide; APADH,

reduced form of APAD; DON, 6-diazo-5-oxo-L-norleucine.

(Received 7 October 2003, revised 2 December 2003,

accepted 23 December 2003)

Eur. J. Biochem. 271, 745–752 (2004)
FEBS 2004 doi:10.1111/j.1432-1033.2003.03973.x