title: 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.
authors.affiliation: Institute for Enzyme Research, Graduate School, College of Agricultural and Life Sciences, University of Wisconsin, Madison 53705, USA.
authors.forename: J D
authors.initials: JD
authors.lastname: Pikus
authors.forename: J M
authors.initials: JM
authors.lastname: Studts
authors.forename: C
authors.initials: C
authors.lastname: Achim
authors.forename: K E
authors.initials: KE
authors.lastname: Kauffmann
authors.forename: E
authors.initials: E
authors.lastname: Münck
authors.forename: R J
authors.initials: RJ
authors.lastname: Steffan
authors.forename: K
authors.initials: K
authors.lastname: McClay
authors.forename: B G
authors.initials: BG
authors.lastname: Fox
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003408:Cresols / Q000378:metabolism; D017931:DNA Primers; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005288:Ferredoxins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010105:Oxygenases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D011549:Pseudomonas / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D015204:Spectroscopy, Mossbauer
chemical_list: D003408:Cresols; D017931:DNA Primers; D005288:Ferredoxins; D011994:Recombinant Proteins; C032538:4-cresol; D010105:Oxygenases; C068610:toluene-4-monooxygenase