Team:Tokyo-NoKoGen/g3dh

About G3DH

The enzyme, glucoside 3-dehydrogenase (E.C.1.1.99.13) or glucose-3-dehydrogenase (G3DH) catalyzes the oxidation of the C-3 hydroxyl group of the glucosides and converting them to corresponding 3-ketoglucosides (1). Because G3DH has wide substrate specificity, it can convert not only monosaccharides but disaccharides, including trehalose.

G3DH is composed of three subunits: a catalytic subunit, a cytochrome c subunit, and a small subunit. The catalytic subunit has a flavin adenine dinucleotide (FAD) cofactor, and the cytochrome c subunit is bound to the cytoplasmic membrane in the periplasm.

In our project, we used G3DH derived from Rhizobium tumefaciens EHA101, former known as Agrobacterium tumefaciens. The enzymatic activity of G3DH in this microorganism was first reported in 1967 (2). The gene encoding this enzyme was found from a putative proteins as the homolog of G3DH derived from Halomonas sp. α-15 (3), which was reported to convert trehalose to 3,3’dkT (see below). The functional expression of R.tumefaciens derived putative enzyme confirmed that the gene encodes the G3DH complex (unpublished data from Sode labtoratory, TUAT).

About 3,3'-diketotrehalose (3,3'-dkT)

3,3’-dkT is a novel trehalose derivative in which the third hydroxyl group of both glucose moieties are oxidized. It was already reported that 3,3-dkT showed an inhibitory effect toward the trehalose from pig-kidney and Bombyx mori (silkworm)(3).

1.Construction of Biobrick

　G3DH gene were cloned from pTrc99a-G3DH which originated from Agrobacterium tumefaciens. PCR products were inserted pSB1C3. Original G3DH gene has two illegal restriction sites. In order to remove these restriction sites, the G3DH gene were amplified by overlap extension PCR. We designed two primer sets for overlap extension PCR.

Fig.1. Remove illegal restriction sites

　G3DH gene fragments were amplified and three PCR products were connected.

　G3DH (removed illegal restriction sites) were ligated with four promoters and double terminator (BBa_B0010 and BBa_B0012).

　One of those promoters is arabinose inducible, and the others are constitutive promoter.

Table 1. Promoter

Fig 2. Constructed plasmid

2. Evaluation

This G3DH needs the maturation of cytochrome c subunit to have catalytic activity. Therefore, we transformed G3DH with the plasmid, pEC86; which expresses cytochrome c maturation (CCM) enzymes (3).

This is the method of culturing and extraction of production (Fig. 3).

We cultured E. coli TOP10 transformed with two plasmids; G3DH expression vector, and cytochrome c maturation enzymes expression vector (pEC86) in LB medium containing 20 mM Trehalose. For expression of G3DH, we used four different promoters.

When OD₆₆₀ achieves 0.6, 0.2 % arabinose was added to the medium for induction in the case we used p_BAD as a promoter.

After culturing for 20 hours at 37℃, we extracted products by boiling and centrifugation.

0.007U trehalase and 20 mM trehalose mixed sample and incubated 30h.

First, we investigated expression of G3DH by SDS-PAGE analysis. Second, we measured the glucose dehydrogenase activity of G3DH. Finally we tried to detect 3,3’-dkT by thin-layer chromatography (TLC).

Fig. 3 evaluation of G3DH

SDS-PAGE

　By SDS-PAGE analysis, there was the band which showed about 68 kDa on G3DH under the constitutive promoter P_medium and P_high, P_BAD. Therefore, we confirmed the expression of G3DH (Fig. 2).

Fig. 4 SDS-PAGE analysis

Measurement of glucose dehydrogenase activity

We measured the oxidase activity of G3DH by PMS-DCIP assay (Fig. 5). On this assay, we measured the decrease in absorbance of DCIP at 600 nm. Substrates were glucose and trehalose. PMS stands for phenazine methosulfate, and DCIP stands for 2,6-dichlorophenol-indophenol.

Thin Layer Chromatography (TLC)

Trehalase inhibition measurement (Fig. )

Fig. 3 Trehalose inhibition activity assay

This is the result of trehalase inhibition activity assay. Each value of activity was normalized at the value of activity of empty vector which were not induced by arabinose. Samples of G3DH which induced by P_high and P_medium activity was lower than that of empty vector.

We concluded that G3DH which induced by P_high and P_medium expressed G3DH. And G3DH converted trehalose to 3,3’-dkT.

Reference

(1) K Kojima et al., (2001) Cloning and Expression of Glucose 3-Dehydrogenase from Halomonas sp. α-15 in Escherichia coli. Biochem Biophys Res Commun, 282, 21-27

(2) K Hayano et al., (1967) Purification und properties of 3-ketosucrose-forming enzyme from the cells of Agrobacterium tumefaciens. J. Biol. Chem.,　242, 3665-3672

(3) K Sode et al., (2001) Enzymatic synthesis of a novel trehalose derivative, 3,3’-diketotrehalose, and its potential application as the trehalase enzyme inhibitor. FEBS Letters, 489, 42-45

(4) E Arslan et al., (1998) Overproduction of the Bradyrhizobium japonicum c-Type Cytochrome Subunits of the cbb3 Oxidase in Escherichia coli. Biochem Biophys Res Commun, 251, 744-747

Ref; http://www.brenda-enzymes.org/enzyme.php?ecno=1.1.99.13