A Novel Pyrroloquinoline Quinone-Dependent 2-Keto- d -Glucose Dehydrogenase from Pseudomonas aureofaciens

Abstract

A gene encoding an enzyme similar to a pyrroloquinoline quinone (PQQ)-dependent sugar dehydrogenase from filamentous fungi, which belongs to new auxiliary activities (AA) family 12 in the CAZy database, was cloned from Pseudomonas aureofaciens. The deduced amino acid sequence of the cloned enzyme showed only low homology to previously characterized PQQ-dependent enzymes, and multiple-sequence alignment analysis showed that the enzyme lacks one of the three conserved arginine residues that function as PQQ-binding residues in known PQQ-dependent enzymes. The recombinant enzyme was heterologously expressed in an Escherichia coli expression system for further characterization. The UV-visible (UV-Vis) absorption spectrum of the oxidized form of the holoenzyme, prepared by incubating the apoenzyme with PQQ and CaCl 2 , revealed a broad peak at approximately 350 nm, indicating that the enzyme binds PQQ. With the addition of 2-keto-D-glucose (2KG) to the holoenzyme solution, a sharp peak appeared at 331 nm, attributed to the reduction of PQQ bound to the enzyme, whereas no effect was observed upon 2KG addition to authentic PQQ. Enzymatic assay showed that the recombinant enzyme specifically reacted with 2KG in the presence of an appropriate electron acceptor, such as 2,6-dichlorophenol indophenol, when PQQ and CaCl 2 were added. 1 H nuclear magnetic resonance ( 1 H-NMR) analysis of reaction products revealed 2-keto-D-gluconic acid (2KGA) as the main product, clearly indicating that the recombinant enzyme oxidizes the C-1 position of 2KG. Therefore, the enzyme was identified as a PQQ-dependent 2KG dehydrogenase (Pa2KGDH). Considering the high substrate specificity, the physiological function of Pa2KGDH may be for production of 2KGA. P yrroloquinoline quinone (PQQ) is a major cofactor in redox enzymes called quinoproteins and was first identified as a cofactor in bacterial methanol dehydrogenase (1) and glucose dehydrogenase (2), in 1979. The presence of PQQ is a defining feature of quinoprotein enzymes, which distinguishes them from nicotinamide-and flavin-dependent enzymes. In nature, PQQ-dependent quinoproteins have primarily been found as bacterial proteins, localized to the periplasm or bound to membranes, which catalyze the oxidation of various sugars and alcohols, such as glucose, methanol, and ethanol, in the presence of an appropriate electron acceptor (3). Because bacterial PQQ-dependent enzymes require cytochrome c or ubiquinone as an electron acceptor, they are believed to be involved in cellular respiration (4, 5).Several Gram-negative bacteria, such as Pseudomonas spp. and Gluconobacter spp., possess a pathway for the oxidation of monosaccharides, known as oxidative fermentation. Among these bacteria, Pseudomonas spp. have been reported to accumulate an oxidized form of glucose in culture (6), producing 2-keto-D-gluconic acid (2KGA) from D-glucose, with D-gluconic acid produced as a metabolic intermediate. The 2KGA biosynthetic pathway proceeds through the sequential catalytic actions of two membrane-bound d...

A Lytic Polysaccharide Monooxygenase with Broad Xyloglucan Specificity from the Brown-Rot Fungus Gloeophyllum trabeum and Its Action on Cellulose-Xyloglucan Complexes

“…For phylogenetic analysis of LPMO9s, amino acid sequences were aligned using MAFFT and then manually edited using Sea View (40). A phylogenetic tree was generated from this alignment by the neighbor-joining method (41) in the ClustalX software (42), with 1,000 bootstraps, as previously described (43). …”

Optimization of an In Vitro Starch Digestibility Assay for Rice

“…Differences between GDHs that arise from their different co‐factors lead to different properties and GDH‐PQQ based glucometers are maltose sensitive whereas GDH‐NAD based glucometers are maltose insensitive . The prosthetic group of GDH‐PQQ differs from that of GDH‐NAD and GDH‐FAD in having a broad substrate specificity to mono‐ and di‐saccharides . The Accu‐Chek Performa contains a mutant Acinobacter calcoaceticus GDH‐PQQ while the FreeStyle Optium Neo utilizes a Pseudomonus sp.…”

“…GDH‐NAD. Although the mechanism behind the sensitivity of GDH‐PQQ to mono‐ and di‐saccharides is not known, Umezawa et al hypothesized that dehydrogenase originated from Pseudomonus sp. specifically oxidizes the C‐1 position of glucose producing gluconic acid while dehydrogenase produced from Acinobacter sp.…”

The Life History of Pyrroloquinoline Quinone (PQQ): A Versatile Molecule with Novel Impacts on Living Systems

“…The extracellular space is acidified due to production of oxidized products and engrossed Ca 2+ in the phosphatic rocks release both H 2 PO 4 and HPO 4 2− in periplasmic space [165]. PQQ acidifies extracellular medium by direct release of acid dissolving mineral phosphate to attain phosphate solubilization ( Figure 16) and P. putida strains with tn5 insertions (Table 6), [166].…”