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Characterization of a gene encoding dihydrolipoamide dehydrogenase of the cyanobacterium Synechocystis sp. strain PCC 6803

Anke Engels

Universität Bielefeld, Biologie VIII: Zellphysiologie, 33501 Bielefeld, Germany

¹ Author for correspondence: Elfriede K. Pistorius. Tel: + 49 521 5601. Fax: + 49 521 6410. e-mail: E.Pistorius@Biologie.Uni-Bielefeid.DE

ABSTRACT

The authors previously reported the isolation and partial characterization of a periplasmically located dihydrolipoamide dehydrogenase (LPD) from the cyanobacterium Synechocystis sp. strain PCC 6803. In the present work the gene (IpdA; database accession number Z48564) encoding the apoprotein of this LPD in Synechocystis PCC 6803 has been identified, sequenced and analysed. The IpdA gene codes for a protein starting with methionine, which is post-translationally removed. The mature protein contains an N-terminal serine and consists of 473 amino acids with a deduced molecular mass of 51421 Da (including one FAD). The LPD is an acidic protein with a calculated isoelectric point of 5.17. Comparison of the amino acid sequence of the Synechocystis LPD with protein sequences in the databases revealed that the enzyme shares identities of 31-35% with all 18 LPDs so far sequenced and published. As a first step in determining the role of this cyanobacterial LPD, attempts were made to generate an LPD-free Synechocystis mutant by insertionally inactivating the IpdA gene with a kanamycin-resistance cassette. However, the selected transformants appeared to be heteroallelic, containing both the intact IpdA gene and the IpdA gene inactivated by the drug-resistance cassette. The heteroallelic mutant studied, which had about 50% of the wild-type LPD activity, caused acidification of the growth medium. Growth over a prolonged time was only possible after an increased buffering of the medium. Since it is reported in the literature that inactivation of the pyruvate dehydrogenase complex (PDC) leads to acidosis, a function of the LPD in a cytoplasmic-membrane-associated PDC is conceivable.

Present address: Universität Tübingen, Mikrobiologie/Biotechnologie, 72076 Tübingen, Germany.

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