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Applied and Environmental Microbiology, July 2006, p. 4853-4861, Vol. 72, No. 7
0099-2240/06/$08.00+0     doi:10.1128/AEM.02758-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Purification and Characterization of Two Enantioselective -Ketoglutarate-Dependent Dioxygenases, RdpA and SdpA, from Sphingomonas herbicidovorans MH

Tina A. Müller,^1, Thomas Fleischmann,¹ Jan Roelof van der Meer,^1,2 and Hans-Peter E. Kohler^1*

Swiss Federal Institute of Aquatic Science and Technology (Eawag), Überlandstrasse 133, CH-8600 Dübendorf, Switzerland,¹ Département de Microbiologie Fondamentale, Bâtiment de Biologie, University of Lausanne, 1015 Lausanne, Switzerland²

Received 22 November 2005/ Accepted 2 May 2006

-Ketoglutarate-dependent (R)-dichlorprop dioxygenase (RdpA) and -ketoglutarate-dependent (S)-dichlorprop dioxygenase (SdpA), which are involved in the degradation of phenoxyalkanoic acid herbicides in Sphingomonas herbicidovorans MH, were expressed and purified as His₆-tagged fusion proteins from Escherichia coli BL21(DE3)(pLysS). RdpA and SdpA belong to subgroup II of the -ketoglutarate-dependent dioxygenases and share the specific motif HXDX₂₄TX₁₃₁HX₁₀R. Amino acids His-111, Asp-113, and His-270 and amino acids His-102, Asp-104, and His 257 comprise the 2-His-1-carboxylate facial triads and were predicted to be involved in iron binding in RdpA and SdpA, respectively. RdpA exclusively transformed the (R) enantiomers of mecoprop [2-(4-chloro-2-methylphenoxy)propanoic acid] and dichlorprop [2-(2,4-dichlorophenoxy)propanoic acid], whereas SdpA was specific for the (S) enantiomers. The apparent K_m values were 99 µM for (R)-mecoprop, 164 µM for (R)-dichlorprop, and 3 µM for -ketoglutarate for RdpA and 132 µM for (S)-mecoprop, 495 µM for (S)-dichlorprop, and 20 µM for -ketoglutarate for SdpA. Both enzymes had high apparent K_m values for oxygen; these values were 159 µM for SdpA and >230 µM for RdpA, whose activity was linearly dependent on oxygen at the concentration range measured. Both enzymes had narrow cosubstrate specificity; only 2-oxoadipate was able to replace -ketoglutarate, and the rates were substantially diminished. Ferrous iron was necessary for activity of the enzymes, and other divalent cations could not replace it. Although the results of growth experiments suggest that strain MH harbors a specific 2,4-dichlorophenoxyacetic acid-converting enzyme, tfdA-, tfdA-, or cadAB-like genes were not discovered in a screening analysis in which heterologous hybridization and PCR were used.

Present address: Microbiology and Molecular Genetics, Michigan State University, 2209 Biomedical Physical Sciences, East Lansing, MI 48824-4320.