A three-component dicamba O-demethylase from Pseudomonas maltophilia, strain DI-6 GENE ISOLATION, CHARACTERIZATION, AND HETEROLOGOUS EXPRESSION

被引:86
作者
Herman, PL [1 ]
Behrens, M [1 ]
Chakraborty, S [1 ]
Chrastil, BM [1 ]
Barycki, J [1 ]
Weeks, DP [1 ]
机构
[1] Univ Nebraska, Dept Biochem, Lincoln, NE 68588 USA
关键词
D O I
10.1074/jbc.M500597200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Dicamba O-demethylase is a multicomponent enzyme from Pseudomonas maltophilia, strain DI-6, that catalyzes the conversion of the widely used herbicide dicamba (2-methoxy-3,6-dichlorobenzoic acid) to DCSA ( 3,6-dichlorosalicylic acid). We recently described the biochemical characteristics of the three components of this enzyme ( i.e. reductase(DIC), ferredoxin(DIC), and oxygenase(DIC)) and classified the oxygenase component of dicamba O-demethylase as a member of the Rieske non-heme iron family of oxygenases. In the current study, we used N-terminal and internal amino acid sequence information from the purified proteins to clone the genes that encode dicamba O-demethylase. Two reductase genes (ddmA1 and ddmA2) with predicted amino acid sequences of 408 and 409 residues were identified. The open reading frames encode 43.7- and 43.9-kDa proteins that are 99.3% identical to each other and homologous to members of the FAD-dependent pyridine nucleotide reductase family. The ferredoxin coding sequence (ddmB) specifies an 11.4-kDa protein composed of 105 residues with similarity to the adrenodoxin family of [2Fe-2S] bacterial ferredoxins. The oxygenase gene (ddmC) encodes a 37.3-kDa protein composed of 339 amino acids that is homologous to members of the Phthalate family of Rieske non-heme iron oxygenases that function as monooxygenases. Southern analysis localized the oxygenase gene to a megaplasmid in cells of P. maltophilia. Mixtures of the three highly purified recombinant dicamba O-demethylase components overexpressed in Escherichia coli converted dicamba to DCSA with an efficiency similar to that of the native enzyme, suggesting that all of the components required for optimal enzymatic activity have been identified. Computer modeling suggests that oxygenaseDIC has strong similarities with the core alpha subunits of naphthalene 1,2-dioxygenase. Nonetheless, the present studies point to dicamba O-demethylase as an enzyme system with its own unique combination of characteristics.
引用
收藏
页码:24759 / 24767
页数:9
相关论文
共 35 条
[1]  
[Anonymous], 1991, Chemistry and Biochemistry of Flavoenzymes
[2]   Genetic analysis of dioxin dioxygenase of Sphingomonas sp. Strain RW1:: Catabolic genes dispersed on the genome [J].
Armengaud, J ;
Happe, B ;
Timmis, KN .
JOURNAL OF BACTERIOLOGY, 1998, 180 (15) :3954-3966
[3]   Molecular characterization of Fdx1, a putidaredoxin-type [2Fe-2S] ferredoxin able to transfer electrons to the dioxin dioxygenase of Sphingomonas sp. RW1 [J].
Armengaud, J ;
Timmis, KN .
EUROPEAN JOURNAL OF BIOCHEMISTRY, 1997, 247 (03) :833-842
[4]   The reductase RedA2 of the multi-component dioxin dioxygenase system of Sphingomonas sp. RW1 is related to class-I cytochrome P450-type reductases [J].
Armengaud, J ;
Timmis, KN .
EUROPEAN JOURNAL OF BIOCHEMISTRY, 1998, 253 (02) :437-444
[5]  
BUNZ PV, 1993, J BACTERIOL, V175, P6467
[6]  
Butler CS, 1997, ADV MICROB PHYSIOL, V38, P47
[7]   A three-component dicamba O-demethylase from Pseudomonas maltophilia, strain DI-6:: Purification and characterization [J].
Chakraborty, S ;
Behrens, M ;
Herman, PL ;
Arendsen, AF ;
Hagen, WR ;
Carlson, DL ;
Wang, XZ ;
Weeks, DP .
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 2005, 437 (01) :20-28
[8]   BIODEGRADATION OF HALOGENATED ORGANIC-COMPOUNDS [J].
CHAUDHRY, GR ;
CHAPALAMADUGU, S .
MICROBIOLOGICAL REVIEWS, 1991, 55 (01) :59-79
[9]  
CORK DJ, 1991, ADV APPL MICROBIOL, V36, P1
[10]   DETECTION, ISOLATION, AND STABILITY OF MEGAPLASMID-ENCODED CHLOROAROMATIC HERBICIDE-DEGRADING GENES WITHIN PSEUDOMONAS SPECIES [J].
CORK, DJ ;
KHALIL, A .
ADVANCES IN APPLIED MICROBIOLOGY, VOL 40, 1995, 40 :289-321