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 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...

Engineering stereoselectivity of ThDP-dependent enzymes

“…Among the enzymes that have been most intensively studied are transketolase (TK) from Escherichia coli and Saccharomyces cerevisiae , acetohydroxy acid synthase (AHAS) isoenzymes I–III from E. coli , benzoylformate decarboxylase (BFD) from Pseudomonas putida , benzaldehyde lyase (BAL) from Pseudomonas fluorescens , pyruvate decarboxylase (PDC) from S. cerevisiae , Zymomonas mobilis and Acetobacter pasteurianus , branched chain ketoacid decarboxylase from Lactococcus lactis , cyclohexane‐1,2‐dione hydrolase from Azoarcus sp. and 2‐succinyl‐5‐enolpyruvyl‐6‐hydroxy‐3‐cyclohexene‐1‐carboxylate (SEPHCHC) synthase (MenD) from E. coli . In addition to MenD, two further α‐ketoglutarate accepting enzymes were studied recently that represent the α‐ketoglutarate decarboxylase part of the α‐ketoglutarate dehydrogenase (KDH) complex and activity: SucA from E. coli and Kgd from Mycobacterium tuberculosis .…”

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

“…Assays contained 100 m m HEPES buffer, pH 8.0, 1 m m NAD + and 0.1 m m CDO. After incubation for 5 min at 37 °C, reactions were started by addition of CDH [18]. Crystals grew at 25 °C by the hanging‐drop method.…”

“…ThDP is strongly anchored to the protein matrix by a divalent metal ion determined as Mg 2+ in CDH by inductively coupled plasma mass spectrometric measurements [18]. Mg 2+ ligates to two phosphate oxygens of the pyrophosphate tail, a water molecule, the main chain oxygen of SerA480 and the side chains of AspA451 and AsnA478, the latter being constituents of the conserved ThDP fingerprint motif ( Fig.…”

“…1). CDH follows Michaelis–Menten kinetics ( k cat,app = 1.6 ± 0.1 s −1 , K M,app = 13.3 ± 0.1 μ m , and k cat,app / K M,app = 1.2 ± 0.1 × 10 5 s −1 · m −1 for CDO, and k cat,app = 1.5 ± 0.1 s −1 , K M,app = 164 ± 31 μ m , k cat,app / K M,app = 0.91 × 10 4 s −1 · m −1 for NAD + ) and contains one ThDP, one Mg 2+ and one FAD molecule per monomer [18] in agreement with the primary structure fingerprints for the ThDP and FAD binding sites [21,22]. As reported for the acetohydroxy acid synthase and glyoxylate carboligase [15,16] the FAD cofactor resists reduction under normal physiological conditions but can be partly reduced with the deazaflavin/oxalate system ( E °′ = −0.65 V).…”

The kinetic characterization and X-ray structure of a putative benzoylformate decarboxylase from M. smegmatis highlights the difficulties in the functional annotation of ThDP-dependent enzymes

“…In both analyses the largest group was the decarboxylase-like (DC) family. While most members of this family are purely decarboxylases, others have evolved to catalyze a variety of redox, hydration, and carboligation reactions [4][5][6]. From an industrial perspective the family is of considerable interest as many members of the DC family, whose in vivo function is the decarboxylation of 2-keto acids, in vitro can catalyze the stereospecific formation of 2-hydroxyketones from two aldehyde molecules [1].…”