Identification, functional characterization, and crystal structure determination of bacterial levoglucosan dehydrogenase

Abstract

Levoglucosan is the 1,6-anhydrosugar of d-glucose formed by pyrolysis of glucans and is found in the environment and industrial waste. Two types of microbial levoglucosan metabolic pathways are known. Although the eukaryotic pathway involving levoglucosan kinase has been well-studied, the bacterial pathway involving levoglucosan dehydrogenase (LGDH) has not been well-investigated. Here, we identified and cloned the gene from the bacterium and characterized the recombinant protein. The enzyme exhibited high substrate specificity toward levoglucosan and NAD for the oxidative reaction and was confirmed to be LGDH. LGDH also showed weak activities (∼4%) toward l-sorbose and 1,5-anhydro-d-glucitol. The reverse (reductive) reaction using 3-keto-levoglucosan and NADH exhibited significantly lower and higher values than those of the forward reaction. The crystal structures of LGDH in the apo and complex forms with NADH, NADH + levoglucosan, and NADH + l-sorbose revealed that LGDH has a typical fold of Gfo/Idh/MocA family proteins, similar to those of-inositol dehydrogenase, aldose-aldose oxidoreductase, 1,5-anhydro-d-fructose reductase, and glucose-fructose oxidoreductase. The crystal structures also disclosed that the active site of LGDH is distinct from those of these enzymes. The LGDH active site extensively recognized the levoglucosan molecule with six hydrogen bonds, and the C3 atom of levoglucosan was closely located to the C4 atom of NADH nicotinamide. Our study is the first molecular characterization of LGDH, providing evidence for C3-specific oxidation and representing a starting point for future biotechnological use of LGDH and levoglucosan-metabolizing bacteria.

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

“…2 ), suggesting that LGDH reaction catalyzed by LgdA is rate-limiting in the in-vitro reconstitution reaction using LG (6 mM) as the substrate. As described above, the LGDH activity of LgdA in reverse reaction (reduction of 3-keto-LG) was higher than that in forward reaction (dehydrogenation of LG), which was consistent with our previous result in LGDH of P. phenanthrenivorans 9 . However, the addition of LgdC caused the marked degradation of LG (Fig.…”

“…LGDH can also oxidize the C3-hydroxyl group of LG, which is the key reaction in the LG metabolic pathway. However, its activity toward d-glucose is very low; 0.05% and 5% activities of LG have been found in Pseudoarthrobacter and Arthrobacter LGDHs, respectively 8,9 , whereas no detectable activity toward d-glucose was observed in S-2701M LGDH in the current study (data not shown). Thus, LgdC catalyzing C3-oxidation of glucose is also thought to be a novel enzyme.…”

“…The region involved in LG metabolism was searched in the draft genome of B. smithii S-2701M (accession no. LC529899) according to the amino acid sequence of LGDH from P. phenanthrenivorans 9 . lgd genes in this region were amplified from the S-2701M genome by PCR using primer sets with 5′-BamHI and 3′-SalI restriction sites for lgdA, lgdB1, and lgdB2, and 5′-EcoRI and 3′-SalI restriction sites for lgdC, respectively.…”

“…Recently, a gene encoding LGDH was found in the genome of Pseudoarthrobacter phenanthrenivorans based on the partial amino acid sequences of the LGDH in Arthrobacter sp. I-552, and the crystal structure of LGDH was determined 9 . The biochemical and structural analyses of LGDH confirmed the C3-specific oxidation of LG that produces 3-keto-LG.…”

“…The biochemical and structural analyses of LGDH confirmed the C3-specific oxidation of LG that produces 3-keto-LG. This finding led us to depict the possible LG metabolic pathway initiated by the LGDH reaction, followed by hydrolysis of 3-keto-LG and reduction of the hydrolysate to produce glucose 5 , 9 . However, no biochemical and genetic analyses have been performed to understand the reactions beyond the dehydrogenation of LG by LGDH in the speculated pathway.…”