Conversion of levoglucosan into glucose by the coordination of four enzymes through oxidation, elimination, hydration, and reduction

Monosaccharide anhydrides such as levoglucosan (LG) are produced during biomass burning and occur widely in the environment, serving as tracers of air pollution. Industrial processes that thermally treat carbohydrates can also generate LG. Two distinct microbial LG utilization pathways were known: a eukaryotic route via LG kinase to glucose-6-phosphate and a bacterial route initiated by an NAD-dependent levoglucosan dehydrogenase (LGDH) that oxidizes LG at C3 to 3-keto-LG. However, steps downstream of LGDH in bacteria had not been biochemically elucidated. Previous work identified an LG-utilizing thermophile, Bacillus smithii S-2701M, and detected glucose formation activity associated with LGDH-containing fractions. The aim of this study was to define the complete bacterial metabolic pathway converting LG to glucose in B. smithii S-2701M by identifying the responsible genes and reconstituting the reactions with recombinant enzymes.

Prior studies established that fungi and yeasts phosphorylate LG via LG kinase to yield glucose-6-phosphate for glycolysis. In bacteria, LGDH activity was identified in Arthrobacter sp. I-552, and subsequently a corresponding gene and crystal structure were reported from Pseudoarthrobacter phenanthrenivorans, confirming specific C3 oxidation of LG to 3-keto-LG. Glycoside hydrolase family 4 (GH4) enzymes are known to catalyze a redox-elimination-hydration-reduction sequence requiring NAD+ and a divalent metal, providing a mechanistic precedent for non-classical glycosidic bond cleavage. Additional relevant literature includes reports on polysaccharide lyases that use β-elimination, and sugar 4,6-dehydratases in SDR superfamily. Limited glucose 3-dehydrogenases (G3DHs) have been described in Cytophaga marinoflava, Halomonas sp., and Agaricus bisporus, typically using non-NAD cofactors. Collectively, these works motivated the search for a bacterial LG pathway comprising oxidation, β-elimination, hydration, and reduction steps.

Genomic analysis of B. smithii S-2701M identified a cluster containing a putative LGDH gene (lgdA) and three adjacent genes (lgdB1, lgdB2, lgdC) encoding predicted sugar-related enzymes (two sugar phosphate isomerase-like proteins and a Gfo/Idh/MocA family oxidoreductase). These four genes were PCR-amplified with restriction sites and cloned into pGEX-6P-1 for expression in Escherichia coli DH5α as GST fusions. Recombinant cultures were grown in LB with ampicillin at 30 °C, induced with 0.1 mM IPTG at OD660 ~0.5, harvested, and lysed. Proteins were purified via glutathione affinity resin with on-column PreScission protease cleavage to remove GST. N-lauroylsarcosine was included during LgdB2 preparation to obtain soluble protein. SDS-PAGE estimated molecular masses: LgdA 44 kDa, LgdB1 30 kDa, LgdB2 36 kDa, LgdC 43 kDa. Enzymatic reactions were reconstituted in vitro and analyzed by TLC (silica gel 60 F254; solvent acetonitrile/water 9:1; detection with 2% 1,3-dihydroxynaphthalene in 95% ethanol). Standard reaction with LG: 50 mM HEPES-NaOH pH 7.5, 0.1–1% LG, 1 mM β-NAD+, 1 mM MnCl2·4H2O, 10 mM 2-mercaptoethanol, recombinant enzymes as indicated; 30 °C for up to 15 h. For 3-keto-LG substrate reactions: 50 mM HEPES-NaOH pH 7.5, 0.3% 3-keto-LG, 1 mM MnCl2·4H2O; β-NADH was added when LgdC was present; 30 °C for 30 min. LGDH and G3DH activities were measured spectrophotometrically at 25 °C monitoring A340 nm for NADH production in 50 mM Tris-HCl with 1 mM β-NAD+ (or NADH as appropriate). Product identification: The LgdB1 product was generated from LG using LgdA + LgdB1 with NAD+ regeneration (31 mM sodium pyruvate and 1 U/mL lactate dehydrogenase), pooled (24 mL), and analyzed by HRMS (ESI-Q-TOF) and NMR (1H 400 MHz, 13C 100 MHz in D2O/H2O 10:90 at 298 K). The LgdB2 product was identified indirectly: the TLC band was extracted rapidly and subjected to the Fukui and Hayano method, measuring UV absorbance in 1 M potassium phosphate buffer pH 7.0 for the diagnostic 310 nm maximum for 3-keto-D-glucose, and by MS (m/z 379.08 for [2M+Na]+). Reverse reaction of LgdC was tested using glucose + NAD+ to produce a TLC spot co-migrating with the LgdB2 product and monitored by NAD+ reduction.

• A four-enzyme pathway converting levoglucosan to glucose in B. smithii S-2701M was reconstituted in vitro using LgdA (LGDH), LgdB1, LgdB2, and LgdC. • LgdA is an NAD-dependent LG dehydrogenase: specific activity 1.32 U/mg with LG (forward reaction) and 6.0 U/mg with 3-keto-LG + NADH (reverse reaction), measured by initial velocity at A340 nm. • Addition of LgdB1 or LgdB2 alleviated product inhibition of LgdA and increased apparent activity. Representative values (specific activity U/mg; A340 after 20 min): none 0.44 ± 0.033 (0.04 ± 0.004); +LgdB1 0.98 ± 0.002 (0.37 ± 0.002); +LgdB2 0.52 ± 0.008 (0.23 ± 0.004); +LgdB1+LgdB2 0.86 ± 0.004 (0.37 ± 0.005); +LgdC alone 0.35 ± 0.037 (0.04 ± 0.003). These data indicate that downstream conversion of 3-keto-LG relieves inhibition and prolongs reaction progression. • LgdB1 catalyzes β-elimination of 3-keto-LG to form 1,5-anhydro-D-erythro-hex-1-en-3-ulose (2-hydroxy-3-keto-D-glucal), detected as a dark green, UV-absorbing TLC spot and confirmed by HRMS and NMR. This reaction did not require Mn2+. • LgdB2 catalyzes hydration of 2-hydroxy-3-keto-D-glucal to 3-keto-D-glucose; Mn2+ markedly enhanced this step. The LgdB2 product exhibited a 310 nm absorbance maximum in phosphate buffer and MS signal at m/z 379.08 ([2M+Na]+), consistent with 3-keto-D-glucose. LgdB2 displayed bifunctionality, showing low-affinity β-elimination of 3-keto-LG in the presence of Mn2+. • LgdC (a Gfo/Idh/MocA family oxidoreductase) reduces 3-keto-D-glucose to D-glucose using NADH. In the reverse direction, LgdC oxidized glucose with NAD+ to yield a product co-migrating with the LgdB2 product on TLC and concomitant NAD+ reduction, supporting its assignment as glucose-3-dehydrogenase in this pathway. • Complete conversion of LG to glucose required NAD+ and Mn2+; 2-mercaptoethanol was dispensable under the conditions tested. LgdB2 alone could partially substitute for LgdB1, consistent with bifunctionality.

The study resolves the previously unknown bacterial steps downstream of LGDH in LG catabolism by demonstrating a coordinated four-enzyme sequence: LgdA-mediated C3 oxidation, LgdB1-catalyzed β-elimination of the 1,6-anhydro bridge, LgdB2-mediated hydration to 3-keto-D-glucose, and LgdC-mediated reduction to glucose. This mechanism mirrors the redox-elimination-hydration-reduction paradigm of GH4 enzymes, which typically execute all steps within a single polypeptide and require NAD+ and a divalent metal. Here, the functions are partitioned across distinct proteins, with Mn2+ particularly important for the hydration step catalyzed by LgdB2. Relief of product inhibition of LgdA by downstream enzymes and NAD+ regeneration via LgdC likely enhance flux through the pathway. The identification of LgdB1 as a 3-keto-LG decyclase (a novel β-elimination acting on an intramolecular 1,6-anhydro linkage), the bifunctional behavior of LgdB2, and the NAD+-utilizing glucose-3-dehydrogenase activity of LgdC highlight new enzyme activities relevant to anhydrosugar metabolism. These findings provide a biochemical basis for bacterial utilization of LG and suggest a mechanistic route to cleave the otherwise recalcitrant 1,6-anhydro linkage by leveraging C3 oxidation-induced lability.

This work elucidates a complete bacterial pathway for converting levoglucosan to glucose in Bacillus smithii S-2701M using four enzymes: LgdA (LGDH), the novel β-elimination enzyme LgdB1, the bifunctional hydrase/β-eliminase LgdB2, and the NAD+-dependent glucose-3-dehydrogenase LgdC. The pathway recapitulates a GH4-like reaction sequence distributed across multiple proteins and requires NAD+ and Mn2+. These insights enable rational engineering of microbial systems for valorizing LG derived from lignocellulosic biomass and suggest B. smithii S-2701M as a promising production host free from catabolite repression on LG. The mechanistic understanding may also inform synthetic routes to anhydrosugars and novel polysaccharides. Future work could expand kinetic characterization, structural elucidation of LgdB1/B2/C, and application of lgd genes in recombinant fermentation platforms for LG conversion.

The LgdB2 reaction product (3-keto-D-glucose) was unstable, preventing direct isolation and full structural characterization; its identification relied on indirect spectral signatures (310 nm absorbance in phosphate buffer) and mass spectrometry of TLC-extracted material. In vitro reconstitution suggested rate limitations at the LGDH step and dependence on cofactors (NAD+, Mn2+), but comprehensive kinetic parameters and in vivo genetic validations within B. smithii S-2701M were not presented. Detailed structural data for the novel enzymes were not provided.