Cyclohexane-1,2-dione hydrolase from denitrifying Azoarcus sp. strain 22Lin, a novel member of the thiamine diphosphate enzyme family

Abstract

Alicyclic compounds with hydroxyl groups represent common structures in numerous natural compounds, such as terpenes and steroids. Their degradation by microorganisms in the absence of dioxygen may involve a C-C bond ring cleavage to form an aliphatic intermediate that can be further oxidized. The cyclohexane-1,2-dione hydrolase (CDH) (EC 3.7.1.11) from denitrifying Azoarcus sp. strain 22Lin, grown on cyclohexane-1,2-diol as a sole electron donor and carbon source, is the first thiamine diphosphate (ThDP)-dependent enzyme characterized to date that cleaves a cyclic aliphatic compound. The degradation of cyclohexane-1,2-dione (CDO) to 6-oxohexanoate comprises the cleavage of a C-C bond adjacent to a carbonyl group, a typical feature of reactions catalyzed by ThDP-dependent enzymes. In the subsequent NAD(+)-dependent reaction, 6-oxohexanoate is oxidized to adipate. CDH has been purified to homogeneity by the criteria of gel electrophoresis (a single band at ∼59 kDa; calculated molecular mass, 64.5 kDa); in solution, the enzyme is a homodimer (∼105 kDa; gel filtration). As isolated, CDH contains 0.8 ± 0.05 ThDP, 1.0 ± 0.02 Mg(2+), and 1.0 ± 0.015 flavin adenine dinucleotide (FAD) per monomer as a second organic cofactor, the role of which remains unclear. Strong reductants, Ti(III)-citrate, Na(+)-dithionite, and the photochemical 5-deazaflavin/oxalate system, led to a partial reduction of the FAD chromophore. The cleavage product of CDO, 6-oxohexanoate, was also a substrate; the corresponding cyclic 1,3- and 1,4-diones did not react with CDH, nor did the cis- and trans-cyclohexane diols. The enzymes acetohydroxyacid synthase (AHAS) from Saccharomyces cerevisiae, pyruvate oxidase (POX) from Lactobacillus plantarum, benzoylformate decarboxylase from Pseudomonas putida, and pyruvate decarboxylase from Zymomonas mobilis were identified as the closest relatives of CDH by comparative amino acid sequence analysis, and a ThDP binding motif and a 2-fold Rossmann fold for FAD binding could be localized at the C-terminal end and central region of CDH, respectively. A first mechanism for the ring cleavage of CDO is presented, and it is suggested that the FAD cofactor in CDH is an evolutionary relict.

Fig. 1.

Degradation of cyclohexane-1,2-diol by Azoarcus sp. strain 22Lin (22). The last two steps are catalyzed by cyclohexane-1,2-dione hydrolase (this work).

Fig. 2.

UV-Vis spectra of the photochemical reduction of CDH (A and B) and reoxidation with K₃[Fe(CN)₆] (C and D) (30 μM CDH, 20 mM Na⁺ oxalate, 3 μM 5-deazaflavin in 50 mM HEPES, pH 8.0). (A) CDH as isolated (solid line) and after 30 s and 4, 12, 17, 22, and 30 min irradiation. (B) Difference spectra of photochemical reduction. (C) Partially reduced CDH (30 min irradiation) and after addition of 7 μM, 48 μM, and 91 μM K₃[Fe(CN)₆]; solid line, CDH fully oxidized as isolated. (D) Difference spectra of reoxidation. The arrows indicate spectral changes upon increase of the irradiation time (A and B) and upon addition of K₃[Fe(CN)₆] (C and D).

Fig. 3.

Forms of cyclohexane-1,2-dione in aqueous solution (2, 36).

Fig. 4.

(A) Dependence of the specific activity of CDH on the concentration of CDO (0.28 μM CDH, 0.5 mM ThDP, 5.0 mM MgSO₄, 1.0 mM NAD⁺, 10 to 500 μM CDO, 0.10 M HEPES, pH 8.0; 37°C; 5 min substrate incubation at 37°C; fit as described in Materials and Methods). (B) Dependence of the specific activity of CDH on the concentration of NAD⁺ (0.47 μM CDH, 0.5 mM ThDP, 5.0 mM MgSO₄, 10 to 1,000 μM NAD⁺, 0.10 M HEPES, pH 8,0; 37°C; 5 min substrate incubation at 37°C; fit as described in Materials and Methods). The error bars indicate standard deviations.

Fig. 5.

Multiple amino acid sequence alignment of ThDP-dependent enzymes. CDH-As, cyclohexane-1,2-dione hydrolase from Azoarcus sp. 22Lin; AHAS-Sc, AHAS from S. cerevisiae (PDB code 1N0H); POX-Lp, POX from L. plantarum (1POW); BFD-Pp, benzoylformate decarboxylase from P. putida (1BFD); and ZPD-Zm, pyruvate decarboxylase from Z. mobilis (1ZPD). The highly conserved N-terminal glutamate and the ThDP binding motif are shaded in red. The green shading indicates the residues of POX-Lp building up β1, α1, β2, and α2 and β3, β4, α3, and β5 of the double Rossmann fold. The residues involved in the potential FAD conserved consensus motif (16) are marked with green arrowheads.

Fig. 6.

Reaction mechanism for the C—C bond ring cleavage of CDO catalyzed by CDH (17), in accordance with the reaction mechanism of POX from L. plantarum (60).