L-Xylo-3-hexulose, a new rare sugar produced by the action of acetic acid bacteria on galactitol, an exception to Bertrand Hudson's rule

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Highlights

  • Galactitol can be oxidized by PQQ-dependent membrane-bound dehydrogenase of Gluconobacter strains and does not conform to well-known Bertrand-Hudson's rule.

  • The oxidized positions were proved to be at the C-3 and C-5 hydroxyl groups of galactitol to produce rare sugars l-xylo-3-hexulose and d-tagatose.

  • This reaction may represent an exception to Bertrand Hudson's rule.

Abstract

Background

In acetic acid bacteria such as Gluconobacter oxydans or Gluconobacter cerinus, pyrroloquinoline quinone (PQQ) in the periplasm serves as the redox cofactor for several membrane-bound dehydrogenases that oxidize polyhydric alcohols to rare sugars, which can be used as a healthy alternative for traditional sugars and sweeteners. These oxidation reactions obey the generally accepted Bertrand Hudson's rule, in which only the polyhydric alcohols that possess cis d-erythro hydroxyl groups can be oxidized to 2-ketoses using PQQ as a cofactor, while the polyhydric alcohols excluding cis d-erythro hydroxyl groups ruled out oxidation by PQQ-dependent membrane-bound dehydrogenases.

Methods

Membrane fractions of G. oxydans were prepared and used as a cell-free catalyst to oxidize galactitol, with or without PQQ as a cofactor.

Results

In this study, we reported an interesting oxidation reaction that the polyhydric alcohols galactitol (dulcitol), which do not possess cis d-erythro hydroxyl groups, can be oxidized by PQQ-dependent membrane-bound dehydrogenase(s) of acetic acid bacteria at the C-3 and C-5 hydroxyl groups to produce rare sugars l-xylo-3-hexulose and d-tagatose.

Conclusions

This reaction may represent an exception to Bertrand Hudson's rule.

General significance

Bertrand Hudson's rule is a well-known theory in polyhydric alcohols oxidation by PQQ-dependent membrane-bound dehydrogenase in acetic acid bacteria. In this study, galactitol oxidation by a PQQ-dependent membrane-bound dehydrogenase represents an exception to the Bertrand Hudson's rule. Further identification of the associated enzymes and deciphering the explicit enzymatic mechanism will prove this theory.

Graphical abstract

Bertrand Hudson's rule is a well-known theory in polyhydric alcohols oxidation by PQQ-dependent membrane-bound dehydrogenase in acetic acid bacteria such as Gluconobacter oxydans. In this study, we found that galactitol, without cis-d-erythro configuration, can also be oxidized by a PQQ-dependent membrane-bound dehydrogenase, represents an exception to the well-known Bertrand Hudson's rule.

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Introduction

Both Gluconobacter and Acetobacter are acetic acid bacteria that belong to the family Acetobacteraceae. Most of the genera of this family are well known as vinegar producers because of their strong ability to oxidize ethanol to acetic acid by membrane-bound dehydrogenases, including alcohol dehydrogenase and aldehyde dehydrogenase. Besides ethanol, Gluconobacter and Acetobacter have a highly active respiratory chain in their membrane, which can oxidize various sugars and sugar alcohols in a stereo- and regio-selective manner to organic acids, aldehydes, and ketones by membrane-bound dehydrogenases [1]. In Gluconobacter oxydans 621H, eight known and two unknown membrane-bound dehydrogenases have been found [2,3]. Most of these membrane-bound dehydrogenases, i.e., glucose dehydrogenase (GOX0265), inositol dehydrogenase (GOX1857), d-sorbitol dehydrogenase (GOX0854-0855), GOX1441, and 0516 are proved to be PQQ-dependent; however, some are also FAD-dependent (i.e., d-gluconate dehydrogenase) [4]. The whole cells of G. oxydans have a higher specific activity of the PQQ-dependent membrane-bound polyol dehydrogenases and can be used as biocatalysts to stereo- and regiospecifically oxidize polyols, primary/secondary alcohols yielding corresponding ketoses or organic acids in high yields [1,5,6].

The stereo- and regiospecificity of Gluconobacter and Acetobacter genera bacterial oxidation in alditol series such as d-arabitol, d-sorbitol, ribitol, glycerol, meso-erythritol, d-mannitol has been expressed as a well-known Bertrand-Hudson rule [7], in which polyols having the d-erythro configuration is oxidized at the secondary alcohol group giving 2-ketose products within the pH range of 5.0–6.5 (Fig. S1a). Whereas alditols without d-erythro configuration, including d-threitol, l-threitol, xylitol, l-arabitol, and dulcitol, excludes oxidation by PQQ-dependent membrane-bound dehydrogenases of A. suboxydans (nowadays as G. suboxydans) [[8], [9], [10], [11]] (Fig. S1b). However, an anomalous oxidation pattern was observed five decades ago in the case of ω-deoxy sugar alcohols such as l-fucitol, a kind of 6-methyl substituted hexitol (Fig. S2a) [8,12]. This reaction to ω-deoxy alcohols was considered as the extension to Bertrand-Hudson rule, in which the terminal methyl group was simply as an elongated CH2OH group, i.e., the methyl group can be seen as the substitution of H atom covalently linked to a carbon atom. Thus the 6-deoxy-l-galactitol has the d-erythro configuration, and it's corresponding –OH group can be oxidized to keto group by A. suboxydans [8] (Fig. S2b).

In this study, we found that the hexitol galactitol (also named as dulcitol) does not possess the cis d-erythro, but d-lyxo configuration can also be acted by genera of Acetobacter sp. and Gluconobacter sp. strains. The enzyme was evidenced to be a PQQ-dependent membrane-bound dehydrogenase. Both of the hydroxyl groups on C-3 and C-5 can be oxidized simultaneously by PQQ-dependent membrane-bound dehydrogenase from Gluconobacter and Acetobacter genera, with l-xylo-3-hexulose (PubChem CID: 18392540, also alternative as d-lyxo-4-hexulose) and d-tagatose as products (Fig. 1). Though d-threitol, xylitol, and d-iditol possess d-lyxo configuration as that of galactitol, however, all these three sugar alcohols cannot be oxidized by PQQ-dependent membrane-bound dehydrogenase from Gluconobacter and Acetobacter genera (Fig. S1b).This galactitol to 3-ketose l-xylo-3-hexulose conversion by PQQ-dependent membrane-bound dehydrogenases may represent an exception to Bertrand Hudson's rule, instead of the extension.

Section snippets

Materials

Sugars or sugar alcohols including galactitol, erythritol, l-erythrulose, d-threitol, xylitol, d-xylulose, d-tagatose, l-tagatose, d-sorbitol, d-arabitol, l-arabitol, l-fucitol, d-mannitol, and nuclease Benzonase were purchased from Merck & Co Inc. or Tokyo Chemical Industry (TCI, Japan). NAD, NADH, NADP, NADPH, PQQ, coomassie brilliant blue R-250, and antibiotics were provided by Sangon Biotech (Shanghai, China). All other reagents or chemicals used were of high-purity grade.

Strains, media, and culture conditions

Microorganisms

Oxidation of galactitol by resting cells of Acetobacter and Gluconobacter strains

The acetic acid bacteria Acetobacter and Gluconobacter are well characterized by their ability to oxidize various sugars and sugar alcohols by cytoplasmic soluble or membrane-bound dehydrogenases using PQQ, FAD, or NAD(P) as cofactors [[1], [2], [3], [4], [5], [6]]. It was reported that galactitol can be oxidized into d-tagatose by G. oxydans [18]. At the first purpose, we screened acetic acid bacteria with the best performance to synthesize d-tagatose from galactitol by the whole-cell

Discussion

In recent years, rare sugars received great interest in nutrition, fine chemicals, and medicine. For instance, d-mannose has recently been found to be an excellent rare sugar that impairs several tumors growth and enhances cell death in response to chemotherapy [25]. d-allose induces programmed cell death in prostate cancer and human retinal progenitor cells (hRPC) by modulating the intrinsic apoptotic pathway [26]. d-tagatose, another important rare sugar, plays a significant role in healthy

Conclusion

Taken together, galactitol is a unique polyol, which not only can be oxidized at C5 to give D-tagatose but also at C3 position to yield another rare ketose L-xylo-3-hexulose by G. oxydans PQQ-dependent membrane-bound dehydrogenase that does not conform to Bertrand-Hudson's rule. Whereas other polyols can only be oxidized at the secondary alcohol group adjacent to the primary terminal alcohol by G. xoydans PQQ-dependent membrane-bound dehydrogenase and obey the Bertrand-Hudson's rule.

Author contributions

HC conceived the conceptualization, funding acquisition, writing the original draft. YX, PC, JL performed the experiments. MB review and edit this manuscript.

Declaration of Competing Interest

The authors declare that they have no competing financial interest.

Acknowledgement

This work was financially supported by the National Natural Science Foundation of China [No. 21877078].

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