Crystal structures of a meta-cleavage product hydrolase from Pseudomonas fluorescens IP01 (CumD) complexed with cleavage products

Abstract

2-Hydroxy-6-oxo-7-methylocta-2,4-dienoate hydrolase (CumD) from Pseudomonas fluorescens IP01 hydrolyzes a meta-cleavage product generated in the cumene (isopropylbenzene) degradation pathway. The crystal structures of the inactive S103A mutant of the CumD enzyme complexed with isobutyrate and acetate ions were determined at 1.6 and 2.0 A resolution, respectively. The isobutyrate and acetate ions were located at the same position in the active site, and occupied the site for a part of the hydrolysis product with CumD, which has the key determinant group for the substrate specificity of related hydrolases. One of the oxygen atoms of the carboxyl group of the isobutyrate ion was hydrogen bonded with a water molecule and His252. Another oxygen atom of the carboxyl group was situated in an oxyanion hole formed by the two main-chain N atoms. The isopropyl group of the isobutyric acid was recognized by the side-chains of the hydrophobic residues. The substrate-binding pocket of CumD was long, and the inhibition constants of various organic acids corresponded well to it. In comparison with the structure of BphD from Rhodococcus sp. RHA1, the structural basis for the substrate specificity of related hydrolases, is revealed.

Fig. 1.

Degradation pathway for cumene (A), toluene (B), ethylbenzene (C), and biphenyl (D). Chemical designations are as follows: compound I, cumene; compound II, 3-isopropylcatechol; compound III, 2-hydroxy-6-oxo-7-methylocta-2,4-dienoate (6-isopropyl-HODA); compound IV, 2-hydroxypenta-2,4-dienoate; compound V, toluene; compound VI, 2-hydroxy-6-oxohepta-2,4-dienoate (6-methyl-HODA); compound VII, ethylbenzene; compound VIII, 2-hydroxy-6-oxoocta-2,4-dienoate (6-ethyl-HODA); compound IX, biphenyl; and compound X, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (6-phenyl-HODA). Enzyme designations are as follows: A, aromatic ring dioxygenase; B, dihydrodiol dehydrogenase; C, extradiol dioxygenase; and D, meta-cleavage compound hydrolase (HODA hydrolase). Compounds III, VI, and VIII serve as good substrates for CumD, whereas compound X does not.

Fig. 2.

Sequence alignment of HODA hydrolases. Multiple sequence alignment was performed using program ClustalX (Thompson et al. 1997) and then modified on the basis of structural alignment. The sequence names are given in blue and red for members of the monoalkylbenzene and biphenyl groups, respectively. The numbering above the alignment is for the CumD sequence. The secondary structures and their designations are shown above CumD and below RHA1 BphD; arrows, large coils, and small coils represent the β-strands, α-helices, and 3₁₀ helices, respectively. Completely conserved residues among the presented sequences are colored red, and conserved residues are shown in pink. The catalytic residues are indicated by red rectangles. The residues involved in the recognition of the 2-hydroxy-6-oxohexa-2,4-dienoate group of the substrate are surrounded by blue frames. The residues involved in the recognition of the carboxylate group and isopropyl group and in the formation of the deeper space of the D-part of CumD are indicated by green, yellow, and orange rectangles, respectively. The residues involved in the recognition of the formation of the D-part of RHA1 BphD are indicated by yellow rectangles. CumD from Pseudomonas fluorescens IP01 (D83955), TodF from P. putida F1 (Y18245), EtbD1 from Rhodococcus sp. RHA1 (AB004320), ThnD from Sphingomonas macrogoltabidus TFA (AF204963), MhpC from Escherichia coli W3110 (D86239), BphD from Burkholderia cepacia LB400 (X66123), CarC from Acrobacterium sp. strain J3 (K. Morii, H. Habe, H. Nojiri, and T. Omori, unpubl.), and BphD from Rhodococcus sp. strain RHA1 (D88016).

Fig. 3.

Superimpositioning of the Cα backbones of CumD and RHA1 BphD. The core and lid domains of CumD are shown in blue and red; those of RHA1 BphD, in light green and green, respectively. The N and C termini are labeled. The isobutyrate ion in the active site (ISB300) and the three catalytic residues of CumD, Ala(Ser)103, Asp224, and His252, are shown as a ball-and-stick model. Root mean square deviation is 1.2 Å, with 245 Cα atoms between type-II ISB and RHA1 BphD (for details, see Materials and Methods).

Fig. 4.

(A) Schematic drawing of the atoms and interactions involved in the recognition of ISB300. (B) Superimpositioning of the sigma-A weighted 2F_o-F_c electron density maps contoured at 2σ and the final model structures of type-II ACT and type-II ISB at the bound acetate and isobutyrate ions. The maps of the type-II ACT and type-II ISB structures are shown in red and blue, respectively. The carbon atoms in the model structures of type-II ACT and type-II ISB are shown in orange and yellow, respectively. Water molecules are represented as green spheres. (C, D) Hydrophobic residues involved in the recognition of the isopropyl group of ISB300. σA-weighted 2F_o-F_c electron density maps contoured at 2σ are shown. The view in D is rotated 90° around the perpendicular axis from that in C.

Fig. 5.

Molecular surfaces of the substrate-binding pockets of CumD (A) and RHA1 BphD (B), calculated using the SPOCK program with probe radius of 1.4 Å. Waters are shown as red spheres. The catalytic serine or mutated alanine residues are labeled in green. The residues involved in the recognition of the isopropyl group of ISB300 and in the formation of the deeper space of the D-part are labeled in yellow and orange, respectively. The residues involved in the formation of the surface of the D-part of RHA1 BphD are labeled in yellow.

Fig. 6.

Modeled substrate in the substrate-binding pocket. The inner surface of the core-domain side (A) and that of the lid-domain side (B) are shown. The side-chain of Ser103 was modeled and energy-minimized with the substrate (see Materials and Methods). The modeled substrate is shown with a ball-and-stick model. The inner surface formed by the residues involved in the catalysis, in the recognition of the carboxylate group and isopropyl group of isobutyric acid, and in the formation of the deeper space of the D-part are colored in red, green, yellow, and orange, respectively. Other conserved residues, which are shown in pink in Fig. 2 ▶, are shown in blue.

Fig. 7.

Superimpositioning of CumD and RHA1 BphD at the D-part. Viewed from a similar direction as in Fig. 3 ▶. Backbone traces are colored as in Fig. 3 ▶. The catalytic residues and the residues involved in the formation of the D-part of CumD are shown as a ball-and-stick model and are labeled in red and black, respectively. The residues of RHA1 BphD are shown and labeled in green.