Molecular cloning and functional expression ofD-sorbitol dehydrogenase fromGluconobacter suboxydansIFO3255, which requires pyrroloquinoline quinone and hydrophobic…

Abstract

The sldA gene that encodes the D-sorbitol dehydrogenase (SLDH) from Gluconobacter suboxydans IFO 3255 was cloned and sequenced. It encodes a polypeptide of 740 residues, which contains a signal sequence of 24 residues. SLDH had 35-37% identity to the membrane-bound quinoprotein glucose dehydrogenases (GDHs) from E. coli, Gluconobacter oxydans, and Acinetobacter calcoaceticus except the N-terminal hydrophobic region of GDH. Additionally, the sldB gene located just upstream of sldA was found to encode a polypeptide consisting of 126 very hydrophobic residues that is similar in sequence to the one-sixth N-terminal region of the GDH. For the development of the SLDH activity in E. coli, co-expression of the sldA and sldB genes and the presence of pyrrloquinolone quinone as a co-factor were required.

Main Polyol Dehydrogenase ofGluconobacter suboxydansIFO 3255, Membrane-boundD-Sorbitol Dehydrogenase, That Needs Product of Upstream Gene,sldB, for Activity

“…The disruptant cells produced D-gluconic acid, so they had D-glucose dehydrogenase activity. The D-glucose dehydrogenase in G. suboxydans IFO 3255 was diŠerent from SLDH, although the amino acid sequence of SLDH 7) was moderately similar to that of D-glucose dehydrogenases. [8][9][10] The diŠerence between SLDH and D-glucose dehydrogenase could be predicted from the substrate speciˆcity of puriˆed SLDH, which is inactive toward D-glucose.…”

“…The gene encoding the membrane-bound SLDH of G. suboxydans IFO 3255, suggested to be a PQQdependent enzyme in our previous study, 7) was disrupted in a check of whether the SLDH was the main dehydrogenase oxidizing D-sorbitol to L-sorbose in strain IFO 3255. In resting-cell reactions, the disruptant 3255sldA::Km did not produce L-sorbose from D-sorbitol at any of the pHs from 4.0 to 8.0, indicating that the SLDH is the main dehydrogenase active toward D-sorbitol whatever the pH.…”

“…We cloned the gene encoding the SLDH of G. suboxydans IFO 3255, sldA, and found that it encoded a polypeptide of 740 amino acid residues including a signal sequence of 24 residues. 7) The polypeptide had 35-37z homology with the membrane-bound and PQQ-dependent D-glucose dehydrogenases of G. oxydans, 8) Acinetobacter calcoaceticus, 9) and Escherichia coli 10) everywhere but the hydrophobic N-termini. We found one open reading frame directly upstream of the gene sldA; this gene, sldB, encoded a polypeptide of 126 amino acid residues that was hydrophobic and had a sequence similar to the hydrophobic N-termini of D-glucose dehydrogenases.…”

“…Plasmid pTNB148 consists of pVK100 and a 0.9-kb SalI fragment containing (i) promoter pA from pUC-pA and (ii) the sldB gene 7) with a stop codon, TGATG, mutagenized to an alternative, TAGTG, to disturb the translation of the sldA gene, the start codon of which begins with the third letter of the stop codon of the sldB gene. The detailed structure of the SalI fragment is shown in Fig.…”

Molecular Properties of Membrane-Bound FAD-ContainingD-Sorbitol Dehydrogenase from ThermotolerantGluconobacter frateuriiIsolated from Thailand

“…In addition, another membrane-bound SLDH (PQQ-SLDH) was recently purified from G. suboxydans var IFO 3255, 5) having one subunit of 80 kDa and pyrroloquinoline quinone (PQQ) as the prosthetic group. Recently, its gene, sldA, composing an operon with sldB, was cloned, 7,8) and the enzyme was shown to be identical to glycerol dehydrogenase, 9) found ubiquitously in Gluconobacter strains, and also to arabitol dehydrogenase 10) and D-gluconate dehydrogenase, yielding 5-keto D-gluconate 11) using molecular biological techniques. The reaction product of this enzyme from Dsorbitol was determined to be L-sorbose, but that of FAD-SLDH has not been determined.…”