Tài liệu Báo cáo khóa học: Tungsten-containing aldehyde oxidoreductase of Eubacterium

Tungsten-containing aldehyde oxidoreductase of Eubacterium

acidaminophilum

Isolation, characterization and molecular analysis

David Rauh, Andrea Graentzdoerffer, Katrin Granderath, Jan R. Andreesen and Andreas Pich*

Institut fu¨r Mikrobiologie, Martin-Luther-Universita¨t Halle-Wittenberg, Halle, Germany

Aldehyde oxidoreductase of Eubacterium acidaminophilum

was purified to homogeneity under strict anaerobic condi￾tions using a four-step procedure. The purified enzyme was

present as a monomer with an apparent molecular mass of

67 kDa and contained 6.0± 0.1 iron, 1.1 ± 0.2 tungsten,

about 0.6 mol pterin cofactor and zinc, but no molybdenum.

The enzyme activity was induced if a molar excess of electron

donors, such as serine and/or formate, were supplied in the

growth medium compared to readily available electron

acceptors such as glycine betaine. Many aldehydes served as

good substrates, thus enzyme activity obtained with acetal￾dehyde, propionaldehyde, butyraldehyde, isovaleraldehyde

and benzaldehyde differed by a factor of less than two.

Kinetic parameters were determined for all substrates tested.

Oligonucleotides deduced from the N-terminal amino acid

sequence were used to isolate the encoding aorA gene and

adjacent DNA regions. The deduced amino acid sequence of

the aldehyde oxidoreductase exhibited high similarities to

other tungsten-containing aldehyde oxidoreductases from

archaea. Transcription of the aorA gene was monocistronic

and started from a r54-dependent promoter. Upstream of

aorA, the gene aorR is localized whose product is similar to

r54-dependent transcriptional activator proteins and, thus,

AorR is probably involved in the regulation of aorA

expression.

Keywords: aldehyde oxidoreductase; tungsten; pyranopterin

cofactor; transcriptional regulation; Eubacterium acidamino￾philum.

Tungsten is a rather rare element [1] and the element with

the largest mass positively involved in living systems. In

recent years, tungsten-containing enzymes have been puri￾fied from a wide variety of microorganisms [2–6]. The

tungsten-containing aldehyde oxidoreductases (AOR) rep￾resent a family within the group of pyranopterin-containing

molybdo- and tungstoenzymes. In contrast to enzymes of

the dimethyl sulfoxide family, tungsten is always at the

enzymatic active site ligated by two pyranopterin cofactors

and an oxo group in the enzymes of the AOR family [4].

These features separate the enzymes of the AOR family

from those of the aldehyde oxidase-type belonging

to the xanthine oxidase (molybdenum hydroxylase) family

(containing molybdenum ligated to just one pyranopterin￾cofactor and a characteristic sulfido group) as observed for

aldehyde oxidase and from sulfate reducers like Desulfovib￾rio gigas or milk xanthine oxidase [7]. The tungsten￾containing AOR family is subdivided into aldehyde

oxidoreductase/aldehyde dehydrogenase (AOR) [8–13],

formaldehyde oxidoreductase (FOR) [14,15], glyceralde￾hyde-3-phosphate oxidoreductase (GAPOR) [16] and carb￾oxylic acid reductase (CAR) [17,18]. These enzymes have

mainly been isolated from hyperthermophilic archaea like

Pyrococcus furiosus or Thermococcus litoralis and from

acetogenic bacteria like Moorella thermoacetica and Clos￾tridium formicoaceticum. P. furiosus contains additional

tungsten-containing enzymes with high similarities to

AOR. Despite these similarities, the purified protein

WOR4 exhibited none of the known enzymatic activities

with aldehydes [19]. Evident from crystallographic studies of

aldehyde oxidoreductase from P. furiosus, tungsten is

coordinated by two cis-enedithiolate groups of two moly￾bdopterin cofactors [11,15,20]. Besides tungsten, all of these

aldehyde oxidoreductases possess also one [4Fe-4S] cluster

whichisinvolvedinelectrontransferfromthetungsten-binding

pterincofactortoanelectronacceptor,usuallyferredoxin.

The anaerobic Gram-positive bacterium Eubacterium

acidaminophilum degrades amino acids by Stickland reac￾tions [21] and possesses two tungsten-containing formate

dehydrogenases that are highly similar in their biochemical

characteristics and share more than 65% identity in their

primary sequence [6,22]. Formate dehydrogenase-I was

partially purified, whereas formate dehydrogenase-II was

purified to homogeneity. Tungsten and iron, but no

molybdenum, were found in the final fractions of both

enzymes. The presence of a tungsten-dependent aldehyde

oxidoreductase activity was induced by high concentrations

Correspondence to J. R. Andreesen, Institut fu¨r Mikrobiologie,

Kurt-Mothes-Str. 3, 06120 Halle, Germany.

Fax: + 49 345 5527010, Tel.: + 49 345 5526350,

E-mail: [email protected]

Abbreviations: AOR, aldehyde oxidoreductase; FOR, formaldehyde

oxidoreductase; GAPOR, glyceraldehyde-3-phosphate

oxidoreductase.

*Present address: Institut fu¨r Pathologie, MH-Hannover,

Carl-Neuberg-Str. 1, 30625 Hannover, Germany.

Note: The nucleotide sequence data reported here are available in the

GenBank database under accession no. AJ318790.

(Received 20 August 2003, revised 29 October 2003,

accepted 12 November 2003)

Eur. J. Biochem. 271, 212–219 (2004)
FEBS 2003 doi:10.1046/j.1432-1033.2003.03922.x