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Journal of Bacteriology, October 1999, p. 5930-5939, Vol. 181, No. 19
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Activity of the Molybdopterin-Containing Xanthine Dehydrogenase of Rhodobacter capsulatus Can Be Restored by High Molybdenum Concentrations in a moeA Mutant Defective in Molybdenum Cofactor Biosynthesis

Silke Leimkühler,1,dagger Sieglinde Angermüller,1 Günter Schwarz,2 Ralf R. Mendel,2 and Werner Klipp1,*

Ruhr-Universität Bochum, Fakultät für Biologie, Lehrstuhl für Biologie der Mikroorganismen, D-44780 Bochum,1 and Botanisches Institut, Technische Universität Braunschweig, D-38023 Braunschweig,2 Germany

Received 13 May 1999/Accepted 27 July 1999

During the screening for Rhodobacter capsulatus mutants defective in xanthine degradation, one Tn5 mutant which was able to grow with xanthine as a sole nitrogen source only in the presence of high molybdate concentrations (1 mM), a phenotype resembling Escherichia coli mogA mutants, was identified. Unexpectedly, the corresponding Tn5 insertion was located within the moeA gene. Partial DNA sequence analysis and interposon mutagenesis of regions flanking R. capsulatus moeA revealed that no further genes essential for molybdopterin biosynthesis are located in the vicinity of moeA and revealed that moeA forms a monocistronic transcriptional unit in R. capsulatus. Amino acid sequence alignments of R. capsulatus MoeA (414 amino acids [aa]) with E. coli MogA (195 aa) showed that MoeA contains an internal domain homologous to MogA, suggesting similar functions of these proteins in the biosynthesis of the molybdenum cofactor. Interposon mutants defective in moeA did not exhibit dimethyl sulfoxide reductase or nitrate reductase activity, which both require the molybdopterin guanine dinucleotide (MGD) cofactor, even after addition of 1 mM molybdate to the medium. In contrast, the activity of xanthine dehydrogenase, which binds the molybdopterin (MPT) cofactor, was restored to wild-type levels after the addition of 1 mM molybdate to the growth medium. Analysis of fluorescent derivatives of the molybdenum cofactor of purified xanthine dehydrogenase isolated from moeA and modA mutant strains, respectively, revealed that MPT is inserted into the enzyme only after molybdenum chelation, and both metal chelation and Mo-MPT insertion can occur only under high molybdate concentrations in the absence of MoeA. These data support a model for the biosynthesis of the molybdenum cofactor in which the biosynthesis of MPT and MGD are split at a stage when the molybdenum atom is added to MPT.

* Corresponding author. Mailing address: Ruhr-Universität Bochum, Fakultät für Biologie, Lehrstuhl für Biologie der Mikroorganismen, D-44780 Bochum, Germany. Phone: 49 (0)234-700-3100. Fax: 49 (0)234-7094-620. E-mail: werner.klipp{at}ruhr-uni-bochum.de.

dagger Present address: Department of Biochemistry, Duke University Medical Center, Durham, NC 27710.

Journal of Bacteriology, October 1999, p. 5930-5939, Vol. 181, No. 19
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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