Soluble expression and purification of the oxidoreductase component of toluene 4-monooxygenase

Abstract

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 ∼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; quantitiative EPR spectroscopy showed ∼1 mol of the S = ½ 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 μM 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.

Reconstitution of Active Mycobacterial Binuclear Iron Monooxygenase Complex in Escherichia coli

“…4). It was reported that the reductase components of binuclear iron monooxygenases that contain [Fe-S] centers were amenable to inactivation in E. coli cells, particularly under high-level-expression conditions (15,24,32). For example, the reductase component of toluene 4-monooxygenase has been functionally expressed under the control of a weak promoter (32).…”

Structural basis for biomolecular recognition in overlapping binding sites in a diiron enzyme system

“…T4moF cell paste was re-suspended in 25 mM MOPS, pH 7.5, containing 80 mM NaCl and 2% glycerol at a ratio of 1.5 ml per g of wet cell paste 55 . The cell-free extract was prepared as described above for T4moH.…”

The apicomplexan Cryptosporidium parvum possesses a single mitochondrial‐type ferredoxin and ferredoxin:NADP+ reductase system

“…The cofactor flavin in holo‐CpmtFNR was further detected and confirmed by an isocratic chromatography‐based method. Greater than 50% of the recombinant CpmtFNR protein contained the cofactor FAD as determined by comparing the sample peak intensity to a standard curve prepared from authentic FAD as previously reported 34…”

“…For the expression of the CpmtFNR protein, a modified terrific broth containing 20 m M glucose, 2 g/L NaNO 3 , and 50 μg/mL ampicillin was used. After growing bacteria at 37°C until OD 600 reached 0.5–0.6, riboflavin (0.01 m M ) and isopropyl‐β‐D‐thio‐galactoside (IPTG) (0.1 m M ) were added into the medium as previously reported 34. The subsequent overexpression was allowed to continue overnight at 16°C.…”