Tài liệu Báo cáo khoa học: Functional characterization of an orphan cupin protein from Burkholderia

Functional characterization of an orphan cupin

protein from Burkholderia xenovorans reveals a

mononuclear nonheme Fe2+-dependent oxygenase

that cleaves b-diketones

Stefan Leitgeb, Grit D. Straganz and Bernd Nidetzky

Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Austria

Introduction

In terms of their physiological functions, which include

enzymatic catalysis, ligand binding, and the role of

storage proteins, the cupins constitute one of the most

diverse superfamilies of proteins known. They have

been described from all three domains of life [1,2], and

usually occur as metalloproteins. Regardless of their

Keywords

cupin; nonheme iron; oxygenase; X-ray

absorption spectroscopy; b-diketone

cleavage

Correspondence

Bernd Nidetzky, Institute of Biotechnology

and Biochemical Engineering, Graz

University of Technology, Petersgasse 12 ⁄ I,

A-8010 Graz, Austria

Fax: +43 316 873 8434

Tel: +43 316 873 8400

E-mail: [email protected]

(Received 15 October 2008, revised 31 July

2009, accepted 17 August 2009)

doi:10.1111/j.1742-4658.2009.07308.x

Cupins constitute a large and widespread superfamily of b-barrel proteins

in which a mononuclear metal site is both a conserved feature of the struc￾ture and a source of functional diversity. Metal-binding residues are con￾tributed from two core motifs that provide the signature for the

superfamily. On the basis of conservation of this two-motif structure, we

have identified an ORF in the genome of Burkholderia xenovorans that

encodes a novel cupin protein (Bxe_A2876) of unknown function. Recom￾binant Bxe_A2876, as isolated from Escherichia coli cell extract, was a

homotetramer in solution, and showed mixed fractional occupancy of its

16.1 kDa subunit with metal ligands (0.06 copper; 0.11 iron; 0.17 zinc).

Our quest for possible catalytic functions of Bxe_A2876 focused on Cu2+

and Fe2+ oxygenase activities known from related cupin enzymes. Fe2+

elicited enzymatic catalysis of O2-dependent conversion of various b-dike￾tone substrates via a nucleophilic mechanism of carbon–carbon bond

cleavage. Data from X-ray absorption spectroscopy (XAS) support a

five-coordinate or six-coordinate Fe2+ center where the metal is bound by

three imidazole nitrogen atoms at 1.98 A˚ . Results of structure modeling

studies suggest that His60, His62 and His102 are the coordinating residues.

In the ‘best-fit’ model, one or two oxygens from water and a carboxylate

oxygen (presumably from Glu96) are further ligands of Fe2+ at estimated

distances of 2.04 A˚ and 2.08 A˚ , respectively. The three-histidine Fe2+ site

of Bxe_A2876 is compared to the mononuclear nonheme Fe2+ centers of

the structurally related cysteine dioxygenase and acireductone dioxygenase,

which also use a facial triad of histidines for binding of their metal cofac￾tor but promote entirely different substrate transformations.

Abbreviations

ARD, acireductone dioxygenase; CDO, cysteine dioxygenase; Dke1, b-diketone-cleaving dioxygenase; DLS, dynamic light scattering; EXAFS,

extended X-ray absorption fine structure; QDO, quercetin dioxygenase; RgCarb, Rubrivivax gelatinosus acetyl ⁄ propionyl-CoA carboxylase;

SOD, superoxide dismutase; XANES, X-ray absorption near-edge structure; XAS, X-ray absorption spectroscopy.

FEBS Journal 276 (2009) 5983–5997 ª 2009 The Authors Journal compilation ª 2009 FEBS 5983