Identification and characterization of a NADH oxidoreductase involved in phenylacetic acid degradation pathway from Streptomyces peucetius.
Annotation of genome of Streptomyces peucetius revealed a putative phenylacetic acid degradation NADH oxidoreductase. RT-PCR analysis of the gene readily showed notable transcription in its native state. The transcription level of paaE when the host is grown on phenylacetic acid showed increased transcription. paaE was cloned into a pET32a(+) vector to overexpress the protein coupled with fusion tags in Escherichia coli BL21(DE3) and purified by immobilized metal affinity chromatography using His-tag. The flavin released from heat-denatured PaaE was identical to that of authentic FAD in HPLC analysis. The purified protein efficiently reduced p-nitroblue tetrazolium (an electron acceptor) in presence of NADH. Cell growth analysis of S. peucetius in phenylacetic acid evidently revealed its involvement in degradation of phenylacetic acid - a key environmental pollutant.
Niraula
Narayan Prasad
NP
Institute of Biomolecule Reconstruction (iBR), Department of Pharmaceutical Engineering, SunMoon University, #100, Kalsan-ri, Tangjeong-myeon, Asan-si, Chungnam 336-708, Republic of Korea.
Shrestha
Pramod
P
Oh
Tae-Jin
TJ
Sohng
Jae Kyung
JK
D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D020869:Gene Expression Profiling; D005822:Genetic Vectors; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009580:Nitroblue Tetrazolium / Q000378:metabolism; D010648:Phenylacetates / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D010648:Phenylacetates; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D009580:Nitroblue Tetrazolium; D009247:NADH, NADPH Oxidoreductases; C025136:phenylacetic acid