Structural and catalytic differences between two FADH(2)-dependent monooxygenases: 2,4,5-TCP 4-monooxygenase (TftD) from Burkholderia cepacia AC1100 and 2,4,6-TCP 4-monooxygenase (TcpA) from Cupriavidus necator JMP134

Abstract

2,4,5-TCP 4-monooxygenase (TftD) and 2,4,6-TCP 4-monooxygenase (TcpA) have been discovered in the biodegradation of 2,4,5-trichlorophenol (2,4,5-TCP) and 2,4,6-trichlorophenol (2,4,6-TCP). TcpA and TftD belong to the reduced flavin adenine dinucleotide (FADH(2))-dependent monooxygenases and both use 2,4,6-TCP as a substrate; however, the two enzymes produce different end products. TftD catalyzes a typical monooxygenase reaction, while TcpA catalyzes a typical monooxygenase reaction followed by a hydrolytic dechlorination. We have previously reported the 3D structure of TftD and confirmed the catalytic residue, His289. Here we have determined the crystal structure of TcpA and investigated the apparent differences in specificity and catalysis between these two closely related monooxygenases through structural comparison. Our computational docking results suggest that Ala293 in TcpA (Ile292 in TftD) is possibly responsible for the differences in substrate specificity between the two monooxygenases. We have also identified that Arg101 in TcpA could provide inductive effects/charge stabilization during hydrolytic dechlorination. The collective information provides a fundamental understanding of the catalytic reaction mechanism and the parameters for substrate specificity. The information may provide guidance for designing bioremediation strategies for polychlorophenols, a major group of environmental pollutants.

Keywords: TCP; TcpA; TftD; bioremediation; crystal structure; flavin; monooxygenase.

Figure 1

(a) 2,4,5-TCP 4-monooxygenase (TftD) oxidizes 2,4,5-TCP to 2,5DiCBQ which is reduced to 2,5-DiCHQ. Then, TftD oxidizes the latter to 5-chloro-2-hydroxy-p-benzoquinone, which can be nonenzymatically reduced to 5-chloro-2-hydroxy-p-hydroquinone; (b) 2,4,6-TCP 4-monooxygenase (TcpA) oxidizes 2,4,6-TCP to 2,6-DiCBQ, which is immediately converted to 6-chloro-2-hydroxy-p-benzoquinone by hydrolytic dechorination and reduced to 2,6-DiCHQ (c) TftD oxidizes 2,4,6-TCP to 2,6-DiCBQ, which is chemically reduced to 2,6-DiCHQ.

Figure 2

Ribbon diagram representing the crystal structure of the TcpA subunit. Secondary structure elements have been numbered sequentially as α1-α18 and β1-β14. N and C refer to the N- and C-terminal regions, respectively. α–helices were shown in red and β-strands were shown in green. Arrangement among the four subunits of TcpA was presented as an inset.

Figure 3

Elution profile of TcpA with multi-angle laser light-scattering (MALLS). Elution profile was shown as absorbance versus elution time with a thin black line representing changes in absorbance at 280 nm. The red line indicated the molecular mass calculated from the light scattering ultimately illustrating the tetrameric nature of TcpA.

Figure 4

Flavin Adenine Dinucleotide binding site in TcpA. Autodock positioning of FAD in the active site of TcpA. Molecular surfacing illustrated Van der Waals interactions of FAD surface with active site surface (Inset).

Figure 5

Amino acid sequence and secondary structure alignment of TcpA with other flavin-dependent monooxygenases. Secondary structure elements for TcpA are depicted as red cylinders and green arrows for α-helices and β-strands, respectively. Secondary structural elements for TftD and HpaB are also highlighted in colors (red for α-helices and green for β-strands). Conserved residues among those monooxygenases are shown in boldface. TcpA, 2,4,6-trichlorophenol monooxygenase from Cupriavidus necator JMP134; TftD, chlorophenol 4-monooxygenase; HpaB, 4-hydroxyphenylacetate 3-monooxygenase from T. thermophilus HB8.

Figure 6

(a) TcpA with docked FADH₂ and 2,5-DiCHQ (b) TftD with docked FADH₂ and 2,5-DiCHQ (c) TcpA with docked FADH₂ and 2,6-DiCHQ (d) TftD with FADH₂ and docked 2,6-DiCHQ.

Figure 7

Ribbon diagram structural alignment of TcpA and TftD monomers. The C-α backbone atoms of TcpA (green) and TftD (red) (PDB: 3HWC) were aligned illustrating high similarity in secondary structural elements. This alignment was performed using Open-Source PyMOL™ (version 1.3).

Figure 8

Proposed mechanism of TcpA.