Purification and characterization of two enantioselective alpha-ketoglutarate-dependent dioxygenases, RdpA and SdpA, from Sphingomonas herbicidovorans MH.

Détails

ID Serval
serval:BIB_4D7377327DE3
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Institution
Titre
Purification and characterization of two enantioselective alpha-ketoglutarate-dependent dioxygenases, RdpA and SdpA, from Sphingomonas herbicidovorans MH.
Périodique
Applied and Environmental Microbiology
Auteur⸱e⸱s
Müller T.A., Fleischmann T., van der Meer J.R., Kohler H.P.
ISSN
0099-2240[print], 0099-2240[linking]
Statut éditorial
Publié
Date de publication
2006
Volume
72
Numéro
7
Pages
4853-4861
Langue
anglais
Résumé
Alpha-ketoglutarate-dependent (R)-dichlorprop dioxygenase (RdpA) and alpha-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 alpha-ketoglutarate-dependent dioxygenases and share the specific motif HXDX(24)TX(131)HX(10)R. 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 Km values were 99 microM for (R)-mecoprop, 164 microM for (R)-dichlorprop, and 3 microM for alpha-ketoglutarate for RdpA and 132 microM for (S)-mecoprop, 495 microM for (S)-dichlorprop, and 20 microM for alpha-ketoglutarate for SdpA. Both enzymes had high apparent Km values for oxygen; these values were 159 microM for SdpA and >230 microM 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 alpha-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-, tfdAalpha-, or cadAB-like genes were not discovered in a screening analysis in which heterologous hybridization and PCR were used.
Mots-clé
2,4-Dichlorophenoxyacetic Acid/analogs & derivatives, 2,4-Dichlorophenoxyacetic Acid/chemistry, 2-Methyl-4-chlorophenoxyacetic Acid/analogs & derivatives, 2-Methyl-4-chlorophenoxyacetic Acid/chemistry, Amino Acid Sequence, Dioxygenases/chemistry, Dioxygenases/genetics, Escherichia coli/enzymology, Escherichia coli/genetics, Herbicides/chemistry, Herbicides/metabolism, Ketoglutaric Acids/metabolism, Mixed Function Oxygenases/metabolism, Molecular Sequence Data, Recombinant Fusion Proteins/genetics, Recombinant Fusion Proteins/metabolism, Sphingomonas/enzymology, Sphingomonas/genetics, Stereoisomerism, Substrate Specificity
Pubmed
Web of science
Open Access
Oui
Création de la notice
21/01/2008 13:35
Dernière modification de la notice
20/08/2019 14:02
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