Tài liệu Báo cáo khoa học: Crystal structure of thiamindiphosphate-dependent indolepyruvate

Crystal structure of thiamindiphosphate-dependent indolepyruvate

decarboxylase from Enterobacter cloacae, an enzyme involved

in the biosynthesis of the plant hormone indole-3-acetic acid

Anja Schu¨ tz1

, Tatyana Sandalova2

, Stefano Ricagno2

, Gerhard Hu¨ bner1

, Stephan Ko¨ nig1

and Gunter Schneider2

1

Institute of Biochemistry, Department of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg,

Germany; 2

Division of Molecular Structural Biology, Department of Medical Biochemistry and Biophysics,

Karolinska Institutet, Stockholm, Sweden

The thiamin diphosphate-dependent enzyme indolepyruvate

decarboxylase catalyses the formation of indoleacetaldehyde

from indolepyruvate, one step in the indolepyruvate path￾way of biosynthesis of the plant hormone indole-3-acetic

acid. The crystal structure of this enzyme from Enterobacter

cloacae has been determined at 2.65 A˚ resolution and refined

to a crystallographic R-factor of 20.5% (Rfree 23.6%). The

subunit of indolepyruvate decarboxylase contains three

domains of open a/b topology, which are similar in structure

to that of pyruvate decarboxylase. The tetramer has pseudo

222 symmetry and can be described as a dimer of dimers.

It resembles the tetramer of pyruvate decarboxylase from

Zymomonas mobilis, but with a relative difference of 20
in

the angle between the two dimers. Active site residues are

highly conserved in indolepyruvate/pyruvate decarboxylase,

suggesting that the interactions with the cofactor thiamin

diphosphate and the catalytic mechanisms are very similar.

The substrate binding site in indolepyruvate decarboxylase

contains a large hydrophobic pocket which can accommo￾date the bulky indole moiety of the substrate. In pyruvate

decarboxylases this pocket is smaller in size and allows dis￾crimination of larger vs. smaller substrates. In most pyruvate

decarboxylases, restriction of cavity size is due to replace￾ment of residues at three positions by large, hydrophobic

amino acids such as tyrosine or tryptophan.

Keywords: crystal structure; protein crystallography; pyru￾vate decarboxylase; substrate specificity; thiamin diphos￾phate.

Plant hormones play central roles in the regulation of plant

growth and development. The first plant hormone to be

described was indole-3-acetic acid (IAA), which is synthe￾sized by plants [1,2] and plant-associated bacteria [3,4].

Several pathways for the synthesis of IAA in these

organisms have been described, and most of them start

from L-tryptophan as precursor. One of the tryptophan￾dependent biosynthetic routes to IAA is the indolepyruvic

acid (IPA) pathway. This pathway starts from L-trypto￾phan, and consists of three steps: (a) the conversion of

tryptophan to indole-3-pyruvic acid; (b) the formation of

indole-3-acetaldehyde; and (c) the production of IAA

(Fig. 1). The first step of the pathway is catalysed by

L-tryptophan aminotransferase, a pyridoxal-5-phosphate￾dependent enzyme [5]. The intermediate, IPA, is decarboxy￾lated by the action of indolepyruvate decarboxylase (IPDC)

[6] and the resulting indole-3-acetaldehyde is oxidized by

an aldehyde oxidase to IAA [7].

Genes encoding IPDC from several microorganisms

have been cloned and characterized. These organisms

include Enterobacter cloacae [8], Pantoea agglomerans [9],

Klebsiella aerogenes [10], Azospirillum brasilense [11,12] and

Azospirillum lipoferum [13]. The IPDC genes code for

polypeptides of about 550 amino acids in length, corres￾ponding to a molecular mass of
60 kDa per subunit. The

enzyme from E. cloacae, which has been characterized

biochemically to some extent, has a molecular mass of

240 kDa, suggesting a tetrameric structure in solution [6].

The enzyme is dependent on Mg2+ and thiamin diphos￾phate as cofactors and has a high affinity for the substrate,

1indolepyruvate (KM ¼ 20 lM; [13a]). The amino acid

sequences of IPDC show homology to pyruvate decarb￾oxylases (PDC) with, for instance, 40% identity between

IPDC from E. cloacae and PDC from Klyveromyces lactis,

38% identity to PDC from Saccharomyces cerevisiae

(ScPDC) and 32% identity to PDC from Zymomonas

mobilis (ZmPDC) [8].

Correspondence to G. Schneider, Department ofMedical Biochemistry

and Biophysics, Tomtebodava¨gen 6, Karolinska Institutet,

S-171 77 Stockholm, Sweden.

Fax: +46 8327626, Tel.: +46 87287675,

E-mail: [email protected]

Abbreviations: IPDC, indolepyruvate decarboxylase; PDC, pyruvate

decarboxylase; EcIPDC, indole-pyruvate decarboxylase from Ente￾robacter cloacae; ZmPDC, PDC from Zymomonas mobilis; ScPDC,

PDC from Saccharomyces cerevisiae; IA A , indole-3-acetic acid;

IPA, indolepyruvic acid; ThDP, thiamin diphosphate.

Note: To facilitate comparison, we are using the nomenclature defined

by Muller et al. (1993) [48] to identify the various domains

in ThDP-dependent enzymes.

(Received 19 January 2003, revised 28 March 2003,

accepted 2 April 2003)

Eur. J. Biochem. 270, 2312–2321 (2003) FEBS 2003 doi:10.1046/j.1432-1033.2003.03601.x