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Journal of Bacteriology, April 2004, p. 1945-1958, Vol. 186, No. 7
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.7.1945-1958.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Crystallographic Comparison of Manganese- and Iron-Dependent Homoprotocatechuate 2,3-Dioxygenases

Matthew W. Vetting,1,2,{dagger} Lawrence P. Wackett,1,2 Lawrence Que Jr.,2,3 John D. Lipscomb,1,2 and Douglas H. Ohlendorf1,2*

Department of Biochemistry, Molecular Biology and Biophysics,1 Department of Chemistry,3 Center for Metals in Biocatalysis, University of Minnesota, Minneapolis, Minnesota 554552

Received 25 July 2003/ Accepted 4 December 2003

The X-ray crystal structures of homoprotocatechuate 2,3-dioxygenases isolated from Arthrobacter globiformis and Brevibacterium fuscum have been determined to high resolution. These enzymes exhibit 83% sequence identity, yet their activities depend on different transition metals, Mn2+ and Fe2+, respectively. The structures allow the origins of metal ion selectivity and aspects of the molecular mechanism to be examined in detail. The homotetrameric enzymes belong to the type I family of extradiol dioxygenases (vicinal oxygen chelate superfamily); each monomer has four ß{alpha}ßßß modules forming two structurally homologous N-terminal and C-terminal barrel-shaped domains. The active-site metal is located in the C-terminal barrel and is ligated by two equatorial ligands, H214NE1 and E267OE1; one axial ligand, H155NE1; and two to three water molecules. The first and second coordination spheres of these enzymes are virtually identical (root mean square difference over all atoms, 0.19 Å), suggesting that the metal selectivity must be due to changes at a significant distance from the metal and/or changes that occur during folding. The substrate (2,3-dihydroxyphenylacetate [HPCA]) chelates the metal asymmetrically at sites trans to the two imidazole ligands and interacts with a unique, mobile C-terminal loop. The loop closes over the bound substrate, presumably to seal the active site as the oxygen activation process commences. An "open" coordination site trans to E267 is the likely binding site for O2. The geometry of the enzyme-substrate complexes suggests that if a transiently formed metal-superoxide complex attacks the substrate without dissociation from the metal, it must do so at the C-3 position. Second-sphere active-site residues that are positioned to interact with the HPCA and/or bound O2 during catalysis are identified and discussed in the context of current mechanistic hypotheses.

* Corresponding author. Mailing address: Department of Biochemistry, Molecular Biology and Biophysics, 6-155 Jackson Hall, 321 Church St. S.E., University of Minnesota Medical School, Minneapolis, MN 55455-0347. Phone: (612) 624-8436. Fax: (612) 624-5121. E-mail: ohlen{at}umn.edu.

{dagger} Present address: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461-1602.

Journal of Bacteriology, April 2004, p. 1945-1958, Vol. 186, No. 7
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.7.1945-1958.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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