Catalytic scope of the thiamine-dependent multifunctional enzyme cyclohexane-1,2-dione hydrolase.

The thiamine diphosphate (ThDP)-dependent enzyme cyclohexane-1,2-dione hydrolase (CDH) was expressed in Escherichia coli and purified by affinity chromatography (Ni-NTA). Recombinant CDH showed the same C-C bond-cleavage and C-C bond-formation activities as the native enzyme. Furthermore, we have shown that CDH catalyzes the asymmetric cross-benzoin reaction of aromatic aldehydes and (decarboxylated) pyruvate (up to quantitative conversion, 92-99 % ee). CDH accepts also hydroxybenzaldehydes and nitrobenzaldehydes; these previously have not (or only in rare cases) been known as substrates of other ThDP-dependent enzymes. On a semipreparative scale, sterically demanding 4-(tert-butyl)benzaldehyde and 2-naphthaldehyde were transformed into the corresponding 2-hydroxy ketone products in high yields. Additionally, certain benzaldehydes with electron withdrawing substituents were identified as potential inhibitors of the ligase activity of CDH.

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

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.

Crystal structure of a ring-cleaving cyclohexane-1,2-dione hydrolase, a novel member of the thiamine diphosphate enzyme family.

The thiamine diphosphate (ThDP) dependent flavoenzyme cyclohexane-1,2-dione hydrolase (CDH) (EC 3.7.1.11) catalyses a key step of a novel anaerobic degradation pathway for alicyclic alcohols by converting cyclohexane-1,2-dione (CDO) to 6-oxohexanoate and further to adipate using NAD(+) as electron acceptor. To gain insights into the molecular basis of these reactions CDH from denitrifying anaerobe Azoarcus sp. strain 22Lin was structurally characterized at 1.26 Å resolution. Notably, the active site funnel is rearranged in an unprecedented manner providing the structural basis for the specific binding and cleavage of an alicyclic compound. Crucial features include a decreased and displaced funnel entrance, a semi-circularly shaped loop segment preceding the C-terminal arm and the attachment of the C-terminal arm to other subunits of the CDH tetramer. Its structural scaffold and the ThDP activation is related to that observed for other members of the ThDP enzyme family. The selective binding of the competitive inhibitor 2-methyl-2,4-pentane-diol (MPD) to the open funnel of CDH reveals an asymmetry of the two active sites found also in the dimer of several other ThDP dependent enzymes. The substrate binding site is characterized by polar and non-polar moieties reflected in the structures of MPD and CDO and by three prominent histidine residues (His28, His31 and His76) that most probably play a crucial role in substrate activation. The NAD(+) dependent oxidation of 6-oxohexanoate remains enigmatic as the redox-active cofactor FAD seems not to participate in catalysis, and no obvious NAD(+) binding site is found. Based on the structural data both reactions are discussed.