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Applied and Environmental Microbiology, February 2006, p. 1487-1495, Vol. 72, No. 2
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.2.1487-1495.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Pyrroloquinoline Quinone-Dependent Dehydrogenases from Ketogulonicigenium vulgare Catalyze the Direct Conversion of L-Sorbosone to L-Ascorbic Acid{dagger}

Taro Miyazaki,* Teruhide Sugisawa,{ddagger} and Tatsuo Hoshino§

Department of Applied Microbiology, Nippon Roche Research Center (NRRC), 200 Kajiwara, Kamakura, Kanagawa 247-8530, Japan

Received 24 April 2005/ Accepted 3 December 2005

A novel enzyme, L-sorbosone dehydrogenase 1 (SNDH1), which directly converts L-sorbosone to L-ascorbic acid (L-AA), was isolated from Ketogulonicigenium vulgare DSM 4025 and characterized. This enzyme was a homooligomer of 75-kDa subunits containing pyrroloquinoline quinone (PQQ) and heme c as the prosthetic groups. Two isozymes of SNDH, SNDH2 consisting of 75-kDa and 55-kDa subunits and SNDH3 consisting of 55-kDa subunits, were also purified from the bacterium. All of the SNDHs produced L-AA, as well as 2-keto-L-gulonic acid (2KGA), from L-sorbosone, suggesting that tautomerization of L-sorbosone causes the dual conversion by SNDHs. The sndH gene coding for SNDH1 was isolated and analyzed. The N-terminal four-fifths of the SNDH amino acid sequence exhibited 40% identity to the sequence of a soluble quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus. The C-terminal one-fifth of the sequence exhibited similarity to a c-type cytochrome with a heme-binding motif. A lysate of Escherichia coli cells expressing sndH exhibited SNDH activity in the presence of PQQ and CaCl2. Gene disruption analysis of K. vulgare indicated that all of the SNDH proteins are encoded by the sndH gene. The 55-kDa subunit was derived from the 75-kDa subunit, as indicated by cleavage of the C-terminal domain in the bacterial cells.

* Corresponding author. Mailing address: Department of Genome Antibody Product Research, Chugai Pharmaceutical Co., Ltd., Fuji Gotemba Research Labs, 1-135 Komakado, Gotemba, Shizuoka 412-8513, Japan. Phone: 81-550-87-6715. Fax: 81-550-87-5326. E-mail: miyazakitru{at}chugai-pharm.co.jp.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

{ddagger} Present address: Biotechnology R&D, DSM Nutritional Products Ltd., Bldg. 203/854, CH-4002 Basel, Switzerland.

§ Present address: Tamagawa University Research Institute, 6-1-1 Tamagawa-Gakuen, Machida, Tokyo 194-8610, Japan.

Applied and Environmental Microbiology, February 2006, p. 1487-1495, Vol. 72, No. 2
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.2.1487-1495.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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