Structure and increased thermostability of Rhodococcus sp. naphthalene 1,2-dioxygenase

Abstract

Rieske nonheme iron oxygenases form a large class of aromatic ring-hydroxylating dioxygenases found in microorganisms. These enzymes enable microorganisms to tolerate and even exclusively utilize aromatic compounds for growth, making them good candidates for use in synthesis of chiral intermediates and bioremediation. Studies of the chemical stability and thermostability of these enzymes thus become important. We report here the structure of free and substrate (indole)-bound forms of naphthalene dioxygenase from Rhodococcus sp. strain NCIMB12038. The structure of the Rhodococcus enzyme reveals that, despite a approximately 30% sequence identity between these naphthalene dioxygenases, their overall structures superpose very well with a root mean square deviation of less than 1.6 A. The differences in the active site of the two enzymes are pronounced near the entrance; however, indole binds to the Rhodococcus enzyme in the same orientation as in the Pseudomonas enzyme. Circular dichroism spectroscopy experiments show that the Rhodococcus enzyme has higher thermostability than the naphthalene dioxygenase from Pseudomonas species. The Pseudomonas enzyme has an apparent melting temperature of 55 degrees C while the Rhodococcus enzyme does not completely unfold even at 95 degrees C. Both enzymes, however, show similar unfolding behavior in urea, and the Rhodococcus enzyme is only slightly more tolerant to unfolding by guanidine hydrochloride. Structure analysis suggests that the higher thermostability of the Rhodococcus enzyme may be attributed to a larger buried surface area and extra salt bridge networks between the alpha and beta subunits in the Rhodococcus enzyme.

FIG. 1.

Multiple sequence alignment of the α and β subunits of NDO-P, NDO-R, and BPDO-R. Secondary structure information for NDO-R is shown above the aligned sequences. The shaded residues are completely conserved, and similar residues based on MultAlin (9) are boxed. The conserved residues that coordinate the mononuclear iron (solid circles) and the Rieske iron-sulfur cluster (triangles) are indicated below the alignment. The bridging aspartic acid is indicated by a star.

FIG. 2.

Circular dichroism spectroscopy plots. (A) Spectra for NDO-R in the far-UV region at 25°C before (⧫) and after (⋄) heating of the sample to 95°C. (B) Temperature-induced unfolding curves for NDO-R (⧫) and NDO-P (⋄) at pH 7.8. Cooperative, though irreversible, unfolding of NDO-P suggests an apparent T_m of 55°C. NDO-R is only partially unfolded at 95°C. (C) Guanidine hydrochloride-induced unfolding curves for NDO-R (⧫) and NDO-P (⋄) at pH 7.8 and 25°C. (D) Urea-induced unfolding curves for NDO-R (⧫) and NDO-P (⋄) at pH 7.8 and 25°C.

FIG. 3.

Stereo view of the active site of NDO-R. MPD from the buffer is found bound in the active site of the native enzyme (structure 1), and indole is bound in the substrate-soaked crystal structure (structure 2). Structure 1 is in gray and structure 2 in yellow. Four water molecules found in the cavity of the active site in structure 1 are shown as red spheres. Two of these waters form part of the octahedral coordination of the iron (dashed lines). A hydrogen bond between MPD and T217 (dashed line) suggests that polar groups on the nonhydroxylating ring of the substrates may interact with T217. A 2FoFc omit map for indole contoured at 0.8 sigma is shown in cyan.

FIG. 4.

Interface salt bridges and hydrogen bonds for NDO-P (A and D), NDO-R (B and E), and BPDO-R (C and F), respectively. The small spheres represent nitrogen and oxygen atoms of subunits α_i (red), β_i (yellow), α_i + 1 (green), and β_i + 1 (blue), which interact to form salt bridges and hydrogen bonds (dashed lines).The mononuclear iron belonging to α_i is shown as a sphere in cyan, and the Rieske cluster belonging to α_i + 1 is shown as spheres in purple. NDO-R and BPDO-R have two salt bridge networks (circled areas 1 and 2 in panels B and C) which involve conserved residues from three subunits. NDO-P has one hydrogen bond network (circled area 1 in panel D) which involves residues from three subunits; NDO-R and BPDO-R have two hydrogen bond networks (circled areas 1 and 2 in panels E and F) which involve residues from three subunits. NDO-P is missing network 2 in panel D, the 8- to 10-residue network, which is present in the other two enzymes. NDO-R and BPDO-R in general have larger networks and more widespread interacting pairs.

FIG. 5.

Cartoon showing likely schemes of dissociation of the α₃β₃ hexamer to trimers, dimers, and monomers. The accessible surface area calculated using a 1.4-Å probe with SYBYL (Tripos) is shown in Ų. The percent increase in accessible surface area upon exposure of the interface buried surface area to the solvent is given next to the arrows. All the numbers are in the order NDO-R (NDO-P) [BPDO-R].

FIG. 6.

View of the active site of NDO-P (from PDB id 1EG9) in green, NDO-R (structure 2) in yellow, and BPDO-R (from PDB id 1ULJ) in purple. The figure illustrates the positions of the bound substrate, the active site mononuclear iron, and the side chains of the residues that interact (similarly or differently) with the bound substrate as discussed in the text.

FIG. 7.

NDO-P has an active site cavity (A) that is 1 Å shallower than NDO-R (B). However, indole is still held in the same place next to the mononuclear iron (structure 2) similar to the indole in NDO-P (PDB id 1EG9). The π-π interaction between F307 in NDO-R and the nonhydroxylated ring of indole seemingly prevents indole from burying itself deeper into the cavity.