The metabolism of cyclohexanol by Acinetobacter NCIB 9871.

Acinetobacter NCIB 9871 was isolated by elective culture on cyclohexanol and grows with this compound as sole source of carbon. It displays a restricted growth spectrum, being unable to grow on a wide range of alternative alicyclic alcohols and ketones. Cyclohexanol-grown cells oxidize the growth substrate at a rate of 230 mul of O2/h per mg dry wt with the consumption of 5.65 mumol of O2/mumol substrate. Cyclohexanone is oxidized at a similar rate with the consumption of 4.85 mumol of O2/mumol. 1-Oxa-2-oxocycloheptane and 6-hydroxyhexanoate are both oxidized at the same slow rate of 44 mul of O2/h per mg dry wt and adipate is not oxidized. Studies with cell extracts reveal the presence of inducible dehydrogenases for cyclohexanol, 6-hydroxyhexanoate and 6-oxohexanoate and a monooxygenase, that in conjunction with a lactonase converts cyclohexanone to 6-hydroxyhexanoate. The monooxygenase is therefore presumed to be of the lactone-forming type and the pathway for conversion of cyclohexanol to adipate; cyclohexanol leads to cyclohexanone leads to 1-oxa-2-oxocycloheptane leads to 6-hydroxyhexanoate leads to 6-oxohexanoate leads to adipate; for which key intermediates have been identified chromatographically, is identical with the route for the oxidation of cyclohexanol by Nocardia globerula CL1.     

Lipase-catalyzed polycondensations: effect of substrates and solvent on chain formation,dispersity, and end-group structure

The effects of substrates and solvent on polymer formation, number-average molecular weight (M(n)), polydispersity, and end-group structure for lipase-catalyzed polycondensations were investigated. Diphenyl ether was found to be the preferred solvent for the polyesterification of adipic acid and 1,8-octanediol giving a M(n) of 28 500 (48 h, 70 degrees C). The effect of varying the alkylene chain length of diols and diacids on the molecular weight distribution and the polymer end-group structure was assessed. A series of diacids (succinic, glutaric, adipic, and sebacic acid) and diols (1,4-butanediol, 1,6-hexanediol, and 1,8-octanediol) were polymerized in solution and in bulk. It was found that reactions involving monomers having longer alkylene chain lengths of diacids (sebacic and adipic acid) and diols (1,8-octanediol and 1,6-hexanediol) give a higher reactivity than reactions of shorter chain-length diacids (succinic and glutaric acid) and 1,4-butanediol. The bulk lipase-catalyzed condensation reactions were feasible, but the use of diphenyl ether gave higher M(n) values (42,400 g/mol in 3 days at 70 degrees C). The polydispersity varied little over the conditions studied giving values 相似文献   

Dissimilation of 1,6-hexanediol and 1,8-octanediol by a hydrocarbon-oxidizingPseudomonas

1,6-Hexanediol is dissimilated by restingPseudomonas aeruginosa cells to give 6-hydroxyhexanoate, which is subject to - as well as to -oxidation.-Oxidation leads to 4-hydroxybutyrate, which is further metabolized through succinate. -Oxidation appears to be inhibited in a reversible manner by the diol. This inhibition leads to the accumulation of 6-hydroxyhexanoate in yields of 50, 75% of the 1,6-hexanediol converted.When the diol is exhausted, the -oxidation of 6-hydroxyhexanoate becomes important, if not preponderant without further adaptation of the resting cells.The hexanedioate formed is most probably subject to -oxidation, yielding succinate, which is further metabolized in the tricarboxylic acid cycle.The dissimilation of 1,8-octanediol is not complicated by a dioicacid route. A pathway through a number of -hydroxy acids linked by -oxidation leads to 4-hydroxybutyrate and succinate.Crude extracts of 1,6-hexanediol- and 1,8-octanediol-grown cells contain an inducible NAD-linked 4-hydroxybutyrate dehydrogenase specific for this hydroxy acid. In addition, a constitutive NADP-linked , -diol dehydrogenase was found, which showed optimum activity with 1,6-hexanediol.Shell Research N.V.     

Improved production of adipate with Escherichia coli by reversal of β-oxidation

The linear C₆ dicarboxylic acid adipic acid is an important bulk chemical in the petrochemical industry as precursor of the polymer nylon-6,6-polyamide. In recent years, efforts were made towards the biotechnological production of adipate from renewable carbon sources using microbial cells. One strategy is to produce adipate via a reversed β-oxidation pathway. Hitherto, the adipate titers were very low due to limiting enzyme activities for this pathway. In most cases, the CoA intermediates are non-natural substrates for the tested enzymes and were therefore barely converted. We here tested heterologous enzymes in Escherichia coli to overcome these limitations and to improve the production of adipate via a reverse β-oxidation pathway. We tested in vitro selected enzymes for the efficient reduction of the enoyl-CoA and in the final reaction for the thioester cleavage. The genes encoding the enzymes which showed in vitro the highest activity were then used to construct an expression plasmid for a synthetic adipate pathway. Expression of paaJ, paaH, paaF, dcaA, and tesB in E. coli BL21(DE3) resulted in the production of up to 36 mg/L of adipate after 30 h of cultivation. Beside the activities of the pathway enzymes, the availability of metabolic precursors may limit the synthesis of adipate, providing another key target for further strain engineering towards high-yield production of adipate with E. coli.

     

Biodegradation of cyclohexylamine by Brevibacterium oxydans IH-35A

A bacterial strain capable of growing on cyclohexylamine (CHAM) was isolated by using enrichment and isolation techniques. The strain isolated, strain IH-35A, was classified as a member of the genus Brevibacterium. The results of growth and enzyme studies are consistent with degradation of CHAM via cyclohexanone (CHnone), 6-hexanolactone, 6-hydroxyhexanoate, and adipate. Cell extracts obtained from this strain grown on CHAM contained CHAM oxidase, and the model for CHAM oxidation by this enzyme was similar to the model for deamino oxidation of amine by amine oxidase.     

Degradation of 3-methylpyridine and 3-ethylpyridine by Gordonia nitida LE31

Cells of Gordonia nitida LE31 grown on 3-methylpyridine degraded 3-ethylpyridine without a lag time and vice versa. Cyclic intermediates were not detected, but formic acid was identified as a metabolite. Degradation of levulinic acid was induced in cells grown on 3-methylpyridine and 3-ethylpyridine. Levulinic aldehyde dehydrogenase and formamidase activities were higher in cells grown on 3-methylpyridine and 3-ethylpyridine than in cells grown on acetate. These data indicate that 3-methylpyridine and 3-ethylpyridine were degraded via a new pathway involving C-2-C-3 ring cleavage.     

Linear copolymeric poly(thia-alkanedioates) by lipase-catalyzed esterification and transesterification of 3,3'-thiodipropionic acid and its dimethyl ester with alpha,omega-alkanediols

Linear copolymeric polyesters (polyoxoesters) containing thioether functions [poly(3,3'-thiodipropionic acid-co-alpha,omega-alkanediols)] were formed in good yield by esterification of an equimolar mixture of 3,3'-thiodipropionic acid (4-thiaheptane-1,7-dioic acid) and 1,6-hexanediol (weight average molecular mass, M(W) >600 Da: approximately 81% after 6 h) or 1,12-dodecanediol (M(W) > 900 Da: approximately 90% after 6 h) catalyzed by immobilized lipase B from Candida antarctica (Novozym 435) for up to 336 h in moderate vacuo without a solvent or drying reagent in the reaction mixture. Poly (3,3'-thiodipropionic acid-co-1,6-hexanediol) and poly (3,3'-thiodipropionic acid-co-1,12-dodecanediol) were extracted from the reaction mixtures using tetrahydrofurane and precipitated from tetrahydrofurane-iso-hexane (1:1, v/v) at approximately 0 degrees C. The precipitate of poly(3,3'-thiodipropionic acid-co-1,6-hexanediol) showed a maximum molecular weight of 6 x 10(5) Da corresponding to a M(W) of approximately 24,200 Da and a degree of polymerization of up to 2,150 monomer units. The precipitated poly(3,3'-thiodipropionic acid-co-1,12-dodecanediol) showed a maximum molecular weight of 8 x 10(5) Da corresponding to a M(W) of approximately 27,200 Da and a maximum degree of polymerization of up to 2,200 monomer units. The chemical structures of both polyesters containing thioether functions were confirmed by chemical derivatization and NMR spectrometry. The chemical structures of various low-molecular weight reaction intermediates of the esterification of 3,3'-thiodipropionic acid with 1,6-hexanediol were elucidated by GC-MS.     

Activated sludge degradation of adipic acid esters.

The biodegradability of three aliphatic adipic acid diesters and a 1,3-butylene glycol adipic acid polyester was determined in acclimated, activated sludge systems. Rapid primary biodegradation from 67 to 99+% was observed at 3- and 13-mg/liter feed levels for di-n-hexyl adipate, di(2-ethylhexyl) adipate, and di(heptyl, nonyl) adipate in 24 h. When acclimated, activated sludge microorganisms were employed as the seed for two carbon dioxide evolution procedures, greater than 75% of the theoretical carbon dioxide was evolved for the three diesters and the polyester in a 35-day test period. The essentially complete biodegradation observed in these studies suggests that these esters would not persist when exposed to similar mixed microbial populations in the environment.     

Activated sludge degradation of adipic acid esters.

The biodegradability of three aliphatic adipic acid diesters and a 1,3-butylene glycol adipic acid polyester was determined in acclimated, activated sludge systems. Rapid primary biodegradation from 67 to 99+% was observed at 3- and 13-mg/liter feed levels for di-n-hexyl adipate, di(2-ethylhexyl) adipate, and di(heptyl, nonyl) adipate in 24 h. When acclimated, activated sludge microorganisms were employed as the seed for two carbon dioxide evolution procedures, greater than 75% of the theoretical carbon dioxide was evolved for the three diesters and the polyester in a 35-day test period. The essentially complete biodegradation observed in these studies suggests that these esters would not persist when exposed to similar mixed microbial populations in the environment.     

Adenosine triphosphate-linked control of Pseudomonas aeruginosa glucose-6-phosphate dehydrogenase

Extracts of Pseudomonas aeruginosa (ATCC 7700) cells grown on glucose, gluconate, or glycerol had enzyme activities related to the Entner-Doudoroff pathway. These activities were present in no more than trace amounts when the bacteria were grown on succinate. Fructose-1,6-diphosphate aldolase could not be detected in extracts of the bacteria grown on any of the above carbon sources. Therefore, it appears that P. aeruginosa degrades glucose via an inducible Entner-Doudoroff pathway. The apparent absence of fructose-1,6-diphosphate aldolase in cells growing on succinate suggests that the bacteria can form hexose and pentose phosphates from succinate by an alternate route. d-Glucose-6-phosphate dehydrogenase, a branch-point enzyme of the Entner-Doudoroff pathway, was purified 50-fold from glucose-grown cells. Its molecular weight, estimated by sucrose density gradient centrifugation, was found to be approximately 190,000. The enzyme was strongly inhibited by adenosine triphosphate, guanosine triphosphate, and deoxyguanosine triphosphate, which decreased the apparent binding of glucose-6-phosphate to the enzyme. It is suggested that adenine nucleotide-linked control of glucose-6-phosphate dehydrogenase may regulate the overall catabolism of hexose phosphates and prevent their wasteful degradation under certain conditions requiring gluconeogenesis.     

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 ? 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.     

The metabolism of benzoate by Moraxella species through anaerobic nitrate respiration. Evidence for a reductive pathway.

Moraxella sp. isolated from soil grows anaerobically on benzoate by nitrate respiration; nitrate or nitrite are obligatory electron acceptors, being reduced to molecular N2 during the catabolism of the substrate. This bacterium also grows aerobically on benzoate. 2. Aerobically, benzoate is metabolized by ortho cleavage of catechol followed by the beta-oxoadipate pathway. 3. Cells of Moraxella grown anaerobically on benzoate are devoid of ortho and meta cleavage enzymes; cyclohexanecarboxylate and 2-hydroxycyclohexanecarboxylate were detected in the anaerobic culture fluid. 4. [ring-U-14C]Benzoate, incubated anaerobically with cells in nitrate-phosphate buffer, gave rise to labelled 2-hydroxycyclohexanecarboxylate and adipate. When [carboxy-14C]benzoate was used, 2-hydroxycyclohexanecarboxylate was radioactive but the adipate was not labelled. A decarboxylation reaction intervenes at some stage between these two metabolites. 5. The anaerobic metabolism of benzoate by Moraxella sp. through nitrate respiration takes place by the reductive pathway (Dutton & Evans, 1969). Hydrogenation of the aromatic ring probably occurs via cyclohexa-2,5-dienecarboxylate and cyclohex-1-enecarboxylate to give cyclohexanecarboxylate. The biochemistry of this reductive process remains unclear. 6. CoA thiol esterification of cyclohexanecarboxylate followed by beta-oxidation via the unsaturated and hydroxy esters, would afford 2-oxocyclohexanecarboxylate. Subsequent events in the Moraxella culture differ from those occurring with Rhodopseudomonas palustris; decarboxylation precedes hydrolytic cleavage of the alicyclic ring to produce adipate in the former, whereas in the latter the keto ester undergoes direct hydrolytic fission to pimelate.     

Degradation of endogenous fructose during catabolism of sucrose and mannitol in halophilic archaebacteria

Metabolism of fructose arising endogenously from sucrose or mannitol was studied in halophilic archaebacteria Haloarcula vallismortis and Haloferax mediterranei. Activities of the enzymes of Embden-Meyerhof-Parnas (EMP) pathway, Entner-Doudoroff (ED) pathway and Pentose Phosphate (PP) pathway were examined in extracts of cells grown on sucrose or mannitol and compared to those grown on fructose and glucose. Sucrase and NAD-specific mannitol dehydrogenase were induced only when sucrose or mannitol respectively were the growth substrates. Endogenously arising fructose was metabolised in a manner similar to that for exogenously supplied fructose i.e. a modified EMP pathway initiated by ketohexokinase. While the enzymes for modified EMP pathway viz. ketohexokinase, 1-phosphofructokinase and fructose 1,6-bisphosphate aldolase were present under all growth conditions, their levels were elevated in presence of fructose. Besides, though fructose 1,6-bisphosphatase, phosphohexoseisomerase and glucose 6-phosphate dehydrogenase were present, the absence of 6-phosphogluconate dehydratase precluded routing of fructose through ED pathway, or through PP pathway directly as 6-phosphogluconate dehydrogenase was lacking. Fructose 1,6-bisphosphatase plays the unusual role of a catabolic enzyme in supporting the non-oxidative part of PP pathway. However the presence of constitutive levels of glucose dehydrogenase and 2-keto 3-deoxy 6-phosphogluconate aldolase when glucose or sucrose were growth substrates suggested that glucose breakdown took place via the modified ED pathway.Abbreviations EMP Embden Meyerhof Parnas - ED Entner Doudoroff - PP pentose phosphate - KHK ketohexokinase - 1-PFK 1-phosphofructokinase - PEP-PTS phosphoenolpyruvate phosphotransferase - 6-PFK 6-phosphofructokinase - FBPase fructose 1,6-bisphosphatase - PHI phosphohexoseisomerase - G6P-DH glucose 6-phosphate dehydrogenase - 6PG-DH 6-phosphogluconate dehydrogenase - GAPDH glyceraldehyde 3-phosphate dehydrogenase - FIP fructose 1-phosphate - GSH reduced glutathione - 2-ME -mercaptoethanol - FBP fructose 1,6-bisphosphate - KDPG 2-keto 3-deoxy 6-phosphogluconate - F6P fructose 6-phosphatez     

Metabolic Engineering of a Novel Muconic Acid Biosynthesis Pathway via 4-Hydroxybenzoic Acid in Escherichia coli

cis,cis-Muconic acid (MA) is a commercially important raw material used in pharmaceuticals, functional resins, and agrochemicals. MA is also a potential platform chemical for the production of adipic acid (AA), terephthalic acid, caprolactam, and 1,6-hexanediol. A strain of Escherichia coli K-12, BW25113, was genetically modified, and a novel nonnative metabolic pathway was introduced for the synthesis of MA from glucose. The proposed pathway converted chorismate from the aromatic amino acid pathway to MA via 4-hydroxybenzoic acid (PHB). Three nonnative genes, pobA, aroY, and catA, coding for 4-hydroxybenzoate hydrolyase, protocatechuate decarboxylase, and catechol 1,2-dioxygenase, respectively, were functionally expressed in E. coli to establish the MA biosynthetic pathway. E. coli native genes ubiC, aroF^FBR, aroE, and aroL were overexpressed and the genes ptsH, ptsI, crr, and pykF were deleted from the E. coli genome in order to increase the precursors of the proposed MA pathway. The final engineered E. coli strain produced nearly 170 mg/liter of MA from simple carbon sources in shake flask experiments. The proposed pathway was proved to be functionally active, and the strategy can be used for future metabolic engineering efforts for production of MA from renewable sugars.     

Adaptive Formation of Fatty Acid Activating Enzyme in Chilomonas paramecium1

SYNOPSIS. Chilomonas paramecium contains 2 different fatty acid activating enzymes (FAAE), one of which utilizes acetate as a substrate, while the other catalyzes the reaction with either butyrate or hexanoate. The site of greatest activity of these enzymes was found to be not in the mitochondrion, but in the “soluble” portion of the cell. Synthesis of acetyl FAAE is constitutive; this enzyme is present regardless of the substrate in the growth medium. The synthesis of the butyryl-hexanoyl FAAE is induced by the presence of either of the substrates. The details of induction of the butyryl enzyme in acetate-grown cells, and the de-adaptation of cells grown in butyrate and transferred to acetate, are given. One mole of pyrophosphate is produced for each mole of CoA-SH reacting, (thus establishing the prevalence of the acyl-adenylate pathway in Chilomonas fatty acid activation.     

Incorporation of [14C]Methanol and [14C]Bicarbonate by Hyphomicrobium sp. 53-49 under Various Growth Conditions: Aerobic,Denitrifying and Autotrophic

A study was made of the incorporation of methanol and bicarbonate into the cell constituents of denitrifying or aerobic methanol grown and autotrophic H₂–O₂–CO₂ grown Hyphomicrobium sp. 53-49. Cells were incubated with [¹⁴C]methanol or [¹⁴C]bicarbonate, and the distribution of the radioactivity in the nonvolatile constituents of ethanol extracts of cells was examined. When denitrifying grown cells were incubated with [¹⁴C]methanol, the major part of the radioactivity was fixed to serine as the first stable compound. Aerobic methanol grown cells also fixed [¹⁴C]methanol mainly to serine. These results suggest that methanol grown cells assimilate methanol by the serine pathway. When denitrifying or aerobic methanol grown cells were incubated with [¹⁴C]bicarbonate, malate was mainly observed as a nonvolatile compound in the initial period of the incubation. Autotrophic grown cells also fixed the major part of [¹⁴C]bicarbonate to malate. In this case, phosphoglyceric acid was found in the phosphorylated compounds area.     

Biosynthesis of d-arabinose in Mycobacterium smegmatis: specific labeling from d-glucose

d-Arabinose is a major sugar in the cell wall polysaccharides of Mycobacterium tuberculosis and other mycobacterial species. The reactions involved in the biosynthesis and activation of d-arabinose represent excellent potential sites for drug intervention since d-arabinose is not found in mammalian cells, and the cell wall arabinomannan and/or arabinogalactan appear to be essential for cell survival. Since the pathway involved in conversion of d-glucose to d-arabinose is unknown, we incubated cells of Mycobacterium smegmatis individually with [1-(14)C]glucose, [3,4-(14)C]glucose, and [6-(14)C]glucose and compared the specific activities of the cell wall-bound arabinose. Although the specific activity of the arabinose was about 25% lower with [6-(14)C]glucose than with other labels, there did not appear to be selective loss of either carbon 1 or carbon 6, suggesting that arabinose was not formed by loss of carbon 1 of glucose via the oxidative step of the pentose phosphate pathway, or by loss of carbon 6 in the uronic acid pathway. Similar labeling patterns were observed with ribose isolated from the nucleic acid fraction. Since these results suggested an unusual pathway of pentose formation, labeling studies were also done with [1-(13)C]glucose, [2-(13)C]glucose, and [6-(13)C]glucose and the cell wall arabinose was examined by NMR analysis. This method allows one to determine the relative (13)C content in each carbon of the arabinose. The labeling patterns suggested that the most likely pathway was condensation of carbons 1 and 2 of fructose 6-phosphate produced by the transaldolase reaction with carbons 4, 5, and 6 (i.e., glyceraldehyde 3-phosphate) formed by fructose-1,6 bisphosphate aldolase. Cell-free enzyme extracts of M. smegmatis were incubated with ribose 5-phosphate, xylulose 5-phosphate, and d-arabinose 5-phosphate under a variety of experimental conditions. Although the ribose 5-phosphate and xylulose 5-phosphate were converted to other pentoses and hexoses, no arabinose 5-phosphate (or free arabinose) was detected in any of these reactions. In addition, these enzyme extracts did not convert arabinose 5-phosphate to any other pentose or hexose. In addition, incubation of [(14)C]glucose 6-phosphate and various nucleoside triphosphates (ATP, CTP, GTP, TTP, and UTP) with cytosolic or membrane fractions from the mycobacterial cells did not result in formation of a nucleotide form of arabinose, although other radioactive sugars including rhamnose and galactose were found in the nucleotide fraction. Furthermore, no radioactive arabinose was found in the nucleotide fraction isolated from M. smegmatis cells grown in [(3)H]glucose, nor was arabinose detected in a large-scale extraction of the sugar nucleotide fraction from 300 g of cells. The logical conclusion from these studies is that d-arabinose is probably produced from d-ribose by epimerization of carbon 2 of the ribose moiety of polyprenylphosphate-ribose to form polyprenylphosphate-arabinose, which is then used as the precursor for formation of arabinosyl polymers.     

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.