Soluble expression and purification of the oxidoreductase component of toluene 4-monooxygenase.
Toluene 4-monooxygenase (T4MO) is a member of the bacterial multicomponent monooxygenases, an enzyme family that utilizes a soluble diiron hydroxylase to oxidize a variety of hydrocarbons as the initial step in their metabolism. The hydroxylases obtain reducing equivalents from NAD(P)H via an electron transfer chain that is initiated by an oxidoreductase containing an N-terminal ferredoxin domain and C-terminal flavin- and NAD-binding domains. T4moF, the NADH oxidoreductase of T4MO, was expressed as a soluble protein in Escherichia coli BL21(DE3) from the pUC-derived expression vector pRS205. This vector contains a lac promoter instead of a T7 promoter. A three step purification from the soluble cell lysate yielded approximately 1 mg of T4moF per gram of wet cell paste with greater than 90% purity. The purified protein contained 1 mol of FAD and 2 mol of Fe per mol of T4moF; quantitative EPR spectroscopy showed approximately 1 mol of the S=1/2 signal from the reduced [2Fe-2S] cluster per mol of T4moF. Steady state kinetic analysis of p-cresol formation activity treating T4moF as the variable substrate while all other proteins and substrates were held constant gave apparent K(M-) and apparent k(cat)-values of 0.15 microM and 3.0 s(-1), respectively. This expression system and purification allows for the recovery of the soluble oxidoreductase in yields that facilitate further biochemical and structural characterizations.
Bailey
Lucas J
LJ
Department of Biochemistry, University of Wisconsin, 433 Babcock Drive, Madison, WI 53706-1544, USA.
Elsen
Nathaniel L
NL
Pierce
Brad S
BS
Fox
Brian G
BG
D002851:Chromatography, High Pressure Liquid; D003124:Colorimetry; D004578:Electron Spin Resonance Spectroscopy; D019075:Electrophoresis, Capillary; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis / Q000737:chemistry; D005822:Genetic Vectors; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D006900:Hydroxylation; D007501:Iron / Q000032:analysis / Q000737:chemistry; D007700:Kinetics; D008970:Molecular Weight; D010088:Oxidoreductases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D010105:Oxygenases / Q000737:chemistry*; D010802:Phylogeny; D011401:Promoter Regions, Genetic; D011489:Protein Denaturation; D012995:Solubility; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
D005182:Flavin-Adenine Dinucleotide; D007501:Iron; D010088:Oxidoreductases; D010105:Oxygenases; C068610:toluene-4-monooxygenase