Arabitol Dehydrogenase Membrane Bound

D-Arabitol ^ D-xylulose

Because there is little information about C-5 sugar alcohol oxidation, evidence has been presented confirming that L-ribulose formation, the oxidation product of ribitol, was catalyzed by the action of a membrane-bound PQQ-dependent ribitol dehydrogenase, but not by a cytosolic NAD-dependent ribitol dehydrogenase [50]. Due to the high hydrophobicity and instability of the enzyme for pen-titol oxidation, its solubilization from the membrane and purification remained to be achieved.

Several physiological and catalytic properties of the purified membrane-bound D-arabitol dehydrogenase (ARDH) have been examined [51]. Solubilization of ARDH from the membrane of Gluconobacter suboxydans IFO 3257 was done successfully with Mydol 10. Selection of a favorable detergent, keeping ARDH as the holoenzyme during all the purification steps by the presence of PQQ and Ca2+, and using a buffer system involving acetate buffer supplemented with Ca2+ were necessary to treat the highly hydrophobic and thus labile enzyme. Purification of ARDH was successful after two steps of column chromatography on DEAE-Toyopearl and CM-Toyopearl in the presence of detergent and Ca2+. The purified ARDH was homogeneous and showed a single sedimentation peak in analytical ultracentrifugation at 3.6 S of the apparent sedimentation constant.

Upon SDS-PAGE, ARDH dissociated into two different subunits of 82 kDa (subunit I) and 14 kDa (subunit II), forming a heterodimeric structure. It contained no heme component, unlike ADH IIB, ADH IIG, and ADH III. ARDH did not react with primary alcohols. The enzyme was proved to be a quinoprotein and dissociation of PQQ was detected by HPLC by SDS-treated ARDH. PQQ and ubiquinone-10 (UQi0) were detected in a purified ARDH when enzyme solubilization was done with dodecyl-P-maltoside instead of Triton X-100. More importantly, when the membrane fraction was treated with 20 mmol L-1 EDTA overnight, ARDH activity was lost but the enzyme activity was restored to its original level by the subsequent addition of PQQ and Ca2+.

ARDH from G. suboxydans IFO 3257 was found to be a versatile enzyme for the oxidation of various sugar alcohols to the corresponding oxidation products, such as glycerol to dihydroxyacetone, ribitol to L-ribulose, D-arabitol to D-xylulose, D-sorbitol to L-sorbose, D-mannitol to D-fructose, and, surprisingly, D-gluconate to 5-keto-D-gluconate, according to the Bertrand-Hudson's rule [52] (Fig. 1.4).

Membrane-bound glycerol dehydrogenase (GLDH) from Gluconobacter indus-trius IFO 3260 was reported to have the same wide substrate specificity as ARDH [53]. However, due to the hydrophobicity, GLDH was unstable when purified and thus insufficient data were obtained to compare it with other PQQ-dependent dehydrogenases, although GLDH has been proved to be an enzyme containing PQQ as the coenzyme. ARDH is very similar to GLDH as well as the membrane-bound quinoprotein D-sorbitol dehydrogenase (SLDH) from G. suboxydans IFO 3255 [54] in the following respects: broad substrate specificity to sugar alcohols, absence of heme component in the enzyme, and a molecular mass of 75-80 kDa in SDS-PAGE. It is worth noting that the enzyme catalyzes D-gluconate oxidation to yield 5-keto-D-gluconate, whereas 2-keto-D-gluconate is produced by an FAD-dependent D-gluconate dehydrogenase (GADH).

The oxidation product of D-gluconate with ARDH was identified as 5-keto-D-gluconate, when purified ARDH was used for cyclic oxidation of D-gluconate in the presence of quinol oxidase and ubiquinone Q2 as illustrated in Fig. 1.5. The

D-Sorbitol ch20h hcoh hoch hcoh HCOH



CH2OH L-Sorbose

Substrate D-MannitoI D-Arabitol



CH2OH D-Fructose




CH2OH D-Xylulose Product

D-Gluconate cooh hcoh hoch hcoh HCOH



CH2OH 5-Keto-D-gluconate

Fig. 1.4 Bertrand-Hudson's rule in polyol oxidation.

Fig. 1.5 D-Gluconate oxidation by a coupling reaction consisting of polyol dehydrogenase and quinol oxidase.

Fig. 1.5 D-Gluconate oxidation by a coupling reaction consisting of polyol dehydrogenase and quinol oxidase.

reaction mixture was only reactive with 5-keto-D-gluconate reductase [55] but not with 2-keto-D-gluconate reductase [56], and a stoichiometric amount of 5-keto-D-gluconate was detected in the reaction mixture. Thus, it was concluded that oxidative fermentation of 5-keto-D-gluconate is catalyzed by polyalcohol dehydrogenase and there is no specific 5-keto-D-gluconate-yielding D-gluconate dehydrogenase. Of course, in contrast to what had previously been believed, a cytosolic 5-keto-D-gluconate reductase cannot possibly contribute to the oxidative fermentation producing 5-keto-D-gluconate.

Regarding the enzyme structure, ARDH may be composed of two different subunits. The presence of a small open reading frame, which is very similar to the transmembrane region of m-GDH in E. coli, has been shown to be essential for the expression of active SLDH (Hoshino et al., personal communication). Therefore, as in SLDH, subunit II may be buried in the cytoplasmic membrane anchoring ARDH to the outer surface of the cytoplasmic membrane. Because the purified ARDH had a relatively high Q2 reductase activity of 12.3 units per mg of protein, ARDH seemed to be attached to the cytoplasmic membranes in vivo and to link to their electron transfer chain via ubiquinone (Adachi et al., unpublished data). In acetic acid bacteria, sugar alcohol oxidation by the membrane-bound enzyme is in accordance with Betrand-Hudson's rule (Fig. 1.4), that is to say, according to the generalization by Bertrand, the most favorable configuration for oxidation has the erythro form and R-configuration of two secondary hydroxyl groups adjacent to the primary alcohol group. ARDH oxidized some secondary alcohols, which must have an R-configuration.

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