Regiospecific oxidation of naphthalene and fluorene by toluene monooxygenases and engineered toluene 4‐monooxygenases of Pseudomonas mendocina KR1

Abstract

The regiospecific oxidation of the polycyclic aromatic hydrocarbons naphthalene and fluorene was examined with Escherichia coli strains expressing wildtype toluene 4-monooxygenase (T4MO) from Pseudomonas mendocina KR1, toluene para-monooxygenase (TpMO) from Ralstonia pickettii PKO1, toluene ortho-monooxygenase (TOM) from Burkholderia cepacia G4, and toluene/ortho-xylene monooxygenase (ToMO) from P. stutzeri OX1. T4MO oxidized toluene (12.1+/-0.8 nmol/min/mg protein at 109 microM), naphthalene (7.7+/-1.5 nmol/min/mg protein at 5 mM), and fluorene (0.68+/-0.04 nmol/min/mg protein at 0.2 mM) faster than the other wildtype enzymes (2-22-fold) and produced a mixture of 1-naphthol (52%) and 2-naphthol (48%) from naphthalene, which was successively transformed to a mixture of 2,3-, 2,7-, 1,7-, and 2,6-dihydroxynaphthalenes (7%, 10%, 20%, and 63%, respectively). TOM and ToMO made 1,7-dihydroxynaphthalene from 1-naphthol, and ToMO made a mixture of 2,3-, 2,6-, 2,7-, and 1,7-dihydroxynaphthalene (26%, 22%, 1%, and 44%, respectively) from 2-naphthol. TOM had no activity on 2-naphthol, and T4MO had no activity on 1-naphthol. To take advantage of the high activity of wildtype T4MO but to increase its regiospecificity on naphthalene, seven engineered enzymes containing mutations in T4MO alpha hydroxylase TmoA were examined; the selectivity for 2-naphthol by T4MO I100A, I100S, and I100G was enhanced to 88-95%, and the selectivity for 1-naphthol was enhanced to 87% and 99% by T4MO I100L and G103S/A107G, respectively, while high oxidation rates were maintained except for G103S/A107G. Therefore, the regiospecificity for naphthalene oxidation was altered to practically pure 1-naphthol or 2-naphthol. All four wildtype monooxygenases were able to oxidize fluorene to different monohydroxylated products; T4MO oxidized fluorene successively to 3-hydroxyfluorene and 3,6-dihydroxyfluorene, which was confirmed by gas chromatography-mass spectrometry and 1H nuclear magnetic resonance analysis. TOM and its variant TomA3 V106A oxidize fluorene to a mixture of 1-, 2-, 3-, and 4-hydroxyfluorene. This is the first report of using enzymes to synthesize 1-, 3-, and 4-hydroxyfluorene, and 3,6-dihydroxyfluorene from fluorene as well as 2-naphthol and 2,6-dihydroxynaphthalene from naphthalene.

Enzymatic hydroxylation of aromatic compounds

“…Aromatic di-iron monooxygenases are versatile biocatalysts that hydroxylate, in addition to their "intrinsic substrate(s)", also other aromatic and even alicyclic compounds. Thus, To4MO regioselectively hydroxylates polycyclic aromatic hydrocarbons, in addition to toluene [79]. Toluene/o-xylene monooxygenase (ToMO) from Pseudomonas stutzeri oxidizes dimethylphenols, cresols, benzene, styrene, and naphthalene, which makes it an interesting candidate for bioremediation purposes.…”

Protein Engineering of Toluene Monooxygenases for Synthesis of Chiral Sulfoxides

“…Three of the residues, Ser, Ala, and Asp/Glu, appeared in both enzymes, emphasizing their contribution to improvement in catalysis. It is also worth noting that some of the variants had been found beneficial in previous work (TOM V106A improved TOM activity for both chloroform degradation and naphthalene oxidation [33], T4MO TmoA I100A and I100S improved 4-nitrocatechol synthesis rate, and TmoA I100G enhanced the selectivity toward 2-naphthol formation from naphthalene [15,45]). Subsequently, the current results as well as previous work by (50) indicate together with the work of Sazinsky et al (37) that the residue in this position need not be hydrophobic for TMOs to be efficient catalysts and suggest that a small side chain is not a prerequisite.…”

2,6-Dihydroxynaphthalene in the Synthesis of Naphthophosphacyclophanes

“…[3][4][5][6][7][8] В качестве такого блока нами был выбран 2,6-дигидроксинафталин (1), который применяется в синтезе олиго-и полимеров, [9][10][11] комплексообразовании [12][13][14] и различных биохимических исследованиях. [15][16][17] Наличие мощной π-электронной системы делает возможным использовать 2,6-дигидроксинафталин в качестве электроно-донорной компоненты в супрамолекулярных ансамблях и синтезе антиоксидантных радикальных ловушек. [18][19][20][21][22][23] Очевидно, что введение в циклофановые структуры атома фосфора может значительно расширить круг их функционального использования.…”