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Applied and Environmental Microbiology, September 1999, p. 3774-3779, Vol. 65, No. 9
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Role of Ngamma -Acetyldiaminobutyrate as an Enzyme Stabilizer and an Intermediate in the Biosynthesis of Hydroxyectoine

David Cánovas,1,2,dagger Nuno Borges,2 Carmen Vargas,1 Antonio Ventosa,1 Joaquín J. Nieto,1,* and Helena Santos2

Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad de Sevilla, 41012 Seville, Spain,1 and Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, 2780 Oeiras, Portugal2

Received 7 April 1999/Accepted 8 June 1999

Strain CHR63 is a salt-sensitive mutant of the moderately halophilic wild-type strain Halomonas elongata DSM 3043 that is affected in the ectoine synthase gene (ectC). This strain accumulates large amounts of Ngamma -acetyldiaminobutyrate (NADA), the precursor of ectoine (D. Cánovas, C. Vargas, F. Iglesias-Guerra, L. N. Csonka, D. Rhodes, A. Ventosa, and J. J. Nieto, J. Biol. Chem. 272:25794-25801, 1997). Hydroxyectoine, ectoine, and glucosylglycerate were also identified by nuclear magnetic resonance (NMR) as cytoplasmic organic solutes in this mutant. Accumulation of NADA, hydroxyectoine, and ectoine was osmoregulated, whereas the levels of glucosylglycerate decreased at higher salinities. The effect of the growth stage on the accumulation of solutes was also investigated. NADA was purified from strain CHR63 and was shown to protect the thermolabile enzyme rabbit muscle lactate dehydrogenase against thermal inactivation. The stabilizing effect of NADA was greater than the stabilizing effect of ectoine or potassium diaminobutyrate. A 1H NMR analysis of the solutes accumulated by the wild-type strain and mutants CHR62 (ectA::Tn1732) and CHR63 (ectC::Tn1732) indicated that H. elongata can synthesize hydroxyectoine by two different pathways---directly from ectoine or via an alternative pathway that converts NADA into hydroxyectoine without the involvement of ectoine.

* Corresponding author. Mailing address: Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad de Sevilla, 41012 Seville, Spain. Phone: 34-954-556765. Fax: 34-954-628162. E-mail: jjnieto{at}cica.es.

dagger Present address: Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología, CSIC, Cantoblanco, 28049 Madrid, Spain.

Applied and Environmental Microbiology, September 1999, p. 3774-3779, Vol. 65, No. 9
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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