Recombinant Toluene-4-monooxygenase:  Catalytic and Mössbauer Studies of the Purified Diiron and Rieske Components of a Four-Protein Complex

Abstract

Expression of the tmoA-F gene cluster from Pseudomonas mendocina KRI in Escherichia coli BL21(DE3) produces a catalytically active form of the toluene-4-monooxygenase (T4MO) complex. Here we report the purification and characterization of four soluble proteins required for the in vitro reconstitution of T4MO catalytic activity. These proteins are a diiron hydroxylase (T4MOH), a Riesketype ferredoxin (T4MOC), an effector protein (T4MOD), and an NADH oxidoreductase (T4MOF). The T4MOH component is composed of the tmoA, tmoB, and tmoE gene products [quaternary structure (alpha beta epsilon)2, Mr approximately 220 kDa]. The T4MOA polypeptide contains two copies of the amino acid sequence motif (D/E)X(28-37)DEXRH; the same motif provides all of the protein-derived ligands to the diiron centers of ribonucleotide reductase, the soluble methane monooxygenase, and the stearoyl-ACP delta 9 desaturase. Mössbauer, optical, and EPR measurements show that the T4MOH contains diiron centers and suggest that the diiron center contains hydroxo bridge(s) in the diferric state, as observed for methane monooxygenase. Mössbauer and EPR measurements also show that the T4MOC contains a Rieske-type iron-sulfur center. This assignment is in accord with the presence of the amino acid sequence motif CPHX(15-17)CX2H, which has also been found in the bacterial, chloroplastic, and mitochondrial Rieske proteins as well as the bacterial NADH-dependent cis-dihydrodiol-forming aromatic dioxygenases. While single-turnover catalytic studies confirm the function of the T4MOH as the hydroxylase, the NADH-dependent multiple-turnover hydroxylation activity is increased by more than 100-fold in the presence of the T4MOC, which mediates highly specific electron transfer between the T4MOF and the T4MOH. The T4MOD can be purified as an 11.6 kDa monomeric protein devoid of cofactors or redox-active metal ions; this component is also detected as a substoichiometric consitutent of the purified T4MOH. The rate of the hydroxylation reaction can be mildly stimulated by the further addition of separately purified T4MOD to the T4MOH, implying the formation of a high affinity, catalytically competent complex between these two components. These characterizations define a novel, four-component oxygenase combining elements from the soluble methane oxidation complex of the methanotrophic bacteria and the aromatic hydroxylation complexes of the soil pseudomonads.

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

“…The T4moF pET15b vector was made in a similar manner by incorporation of 5′ NcoI and 3′ BamHI restriction sites. Vector pT4moABEF was made from pRS184f [7] by digestion with KpnI, which removed 531 bp spanning between the tmoc and tmod genes, and re-ligation to generate a non-functional tmoc-tmod fusion.…”

“…These fractions were assayed for activity using the T4MO-catalyzed oxidation of nitrobenzene to p-nitrophenol (described below). Pooled fractions were also assayed for the conversion of toluene to p-cresol by gas chromatography [7].…”

“…The electron transfer activity of T4moF was determined by reconstitution of the hydroxylation activity of the T4MO complex according to established protocols [7]. Individual fractions from the chromatography steps were assayed monitoring the conversion of nitrobenzene to pnitrophenol.…”

“…The pooled fractions were further analyzed for the conversion of toluene to p-cresol using a variation of the previously described method [7]. First, multiple assays were performed with serial dilution of the T4moF fractions in order to assure that the rate of observed product formation was linearly dependent on the amount of T4moF added (addition of excess T4moF would cause an underestimation of the specific activity).…”

“…The hydroxylase (T4moH), effector protein (T4moD) and Rieske-type ferredoxin (T4moC) of the T4MO system have been purified and extensively characterized [2,[5][6][7][8][9][10][11][12][13]. In contrast, study of T4moF has been limited by difficulties in obtaining soluble expression, with subsequent limits on the ability to obtain purified protein.…”