Identification of a novel 4-hydroxyphenylpyruvate dioxygenase from the soil metagenome.
4-Hydroxyphenylpyruvate dioxygenase (HPPD) is a Fe(II)-dependent, non-heme oxygenase that converts 4-hydroxyphenylpyruvate to homogentisate. Essential cofactors, such as plastoquinone and tocopherol, are produced by HPPD-dependent anabolic pathways in plants. To isolate a novel hppd using culture-independent method, a cosmid metagenomic library was constructed from soil in Korea. Screening of Escherichia coli metagenomic libraries led to the identification of a positive clone, YS103B, producing dark brown pigment in Luria-Bertani medium supplemented with l-tyrosine. In vitro transposon mutagenesis of YS103B showed that the 1.3kb insert was sufficient to produce the hemolytic brown pigment. Sequence analysis of YS103B disclosed one open reading frame encoding a 41.4kDa protein with the well-conserved prokaryotic oxygenase motif of the HPPD family of enzymes. The HPPD-specific beta-triketone herbicide, sulcotrione, inhibited YS103B pigmentation. The recombinant protein expressed in E. coli generated homogentisic acid. Thus, we present the successful heterologous expression of a previously uncharacterized hppd gene from an uncultured soil bacterium.
Lee
Chang-Muk
CM
Microbial Genetics Division, National Institute of Agricultural Biotechnology, 224, Suinro, Rural Development Administration, Suwon 441-707, Republic of Korea.
Yeo
Yun-Soo
YS
Lee
Jung-Han
JH
Kim
Soo-Jin
SJ
Kim
Jung-Bong
JB
Han
Nam Soo
NS
Koo
Bon-Sung
BS
Yoon
Sang-Hong
SH
D015111:4-Hydroxyphenylpyruvate Dioxygenase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003512:Cyclohexanones / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D016680:Genome, Bacterial; D015698:Genomic Library; D006540:Herbicides / Q000494:pharmacology; D008698:Mesylates / Q000494:pharmacology; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification; D020539:Sequence Analysis, Protein; D012988:Soil Microbiology
D001426:Bacterial Proteins; D003512:Cyclohexanones; D006540:Herbicides; D008698:Mesylates; D011994:Recombinant Proteins; C079906:sulcotrione; D015111:4-Hydroxyphenylpyruvate Dioxygenase