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 COC 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 COC 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 ϳ59 kDa; calculated molecular mass, 64.5 kDa); in solution, the enzyme is a homodimer (ϳ105 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.Alicyclic compounds, such as steroids and terpenes, are widespread in nature. They are produced by plant cells as secondary metabolites and occur in fossil fuels. Microorganisms can convert these compounds to cellular metabolites under oxic and anoxic conditions. Their biodegradation proceeds via COC bond ring cleavage to form an aliphatic intermediate, which can be further degraded by ␤-oxidation. In aerobic bacteria, the cleavage of the cyclic compound is catalyzed by a NADPH-dependent, flavin-containing monooxygenase. For example, cyclohexanone is converted to ε-caprolactone in a Bayer-Villiger-type reaction (14). Subsequently, the lactone is hydrolyzed to 6-hydroxyhexanoate (63), followed by two NAD ϩ /NADP ϩ -dependent oxidation steps with adipate as the final product. In anaerobes, such as Pseudomonas sp. strain K601, cyclohexa...

Alicyclic alcohols are naturally occurring compounds wh ich can be degraded by microorgan isms via cleavage of the ring CC bond. DenitrifyingAzoarcus sp. strain 22Lin grows on cyclohexane-l .2-diol which serves as electron donor and carbon source. The diol is converted to cyclohexane-l.2-dione followed by hydrolysis to the corresponding semialdehyde and oxidation to adipate. The latter two reactions are catalyzed by the thiamine diphosphate-dependent llavoenzyme cyciohexane-1.2-dione hydrolase. the first a-ketolase known so far. Biochemical and structural properties of this new member of the thiamine diphosphate enzyme family will be presented.

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 semicircularly 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. Abbreviations CDH, cyclohexane-1,2-dione hydrolase; CDO, cyclohexane-1,2-dione; MPD, 2-methyl-2,4-pentane-diol; ThDP, thiamine diphosphate.

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