New Insights into the Molecular Enzymology of Pyruvate Metabolism in the Halophilic Archaea
David W HOUGH*, Deborah G. MADDOCKS, and Michael J. DANSON
Centre for Extremophile Research, Department of Biology & Biochemistry, University of Bath, Bath BA2 7AY, UK
In the Eukarya and most aerobic Bacteria, pyruvate is converted to acetyl-CoA via the pyruvate dehydrogenase multienzyme complex [PDHC]. This complex is a member of the 2-oxoacid dehydrogenase complex family, and is a three-component system consisting of enzymes E 1 [pyruvate decarboxylase], E2 [lipoate acetyl- transferase] and E3 [dihydrolipoamide dehydrogenase]. In contrast, the Archaea catalyse this reaction via a simpler pyruvate ferredoxin oxidoreductase, and until now no PDHC activity has been found in this Domain.
We have previously discovered the presence of dihydrolipoamide dehydrogenase [DHLipDH] and its presumed substrate, lipoic acid, inthe halophilic Archaea (1). The enzyme catalyses the reaction:
Dihydrolipoamide + NAD+ ―――→ lipoamide + NADH + H+
Given that the only known function of DHLipDH and lipoic acid in non-archaeal organisms is in the 2-oxoacid dehydrogenase complexes, and that these multienzyme complexes are unknown in the Archaea, the discovery of DHLipDH in the halophiles was a surprise and no function has yet been ascribed to this archaeal enzyme.
The gene encoding Haloferax volcanii DHLipDH has been cloned and sequenced (2), and this presentation will describe:
・ Homologous expression of the H. volcanii DHLipDH, and characterisation of the recombinant enzyme (3).
・ The creation and characterisation of a DHLipDH-minus strain of H. volcanii.
・ The generation of a homology-modelled structure of the halophilic DHLipDH, and rational site-directed mutagenesis to explore its halophilic properties(4).
・ The presence of the H. volcanii DHLipDH gene in an operon that contains genes homologous to E 1 and E2 components of non-archaeal 2-oxoacid dehydrogenase complexes, and most closely to those of bacterial PDHC.
This is the first report of the possible presence of the PDHC multienzyme system in an Archaeon, and the metabolic and evolutionary significance of this discovery will be discussed.
1. Danson, M.J., Hough, D.W., Vettakkommakankav, N.N. and Stevenson, K.J. (1991) NATO ASI Series A, 201. 121 - 128.
2. Vettakkommakankav, N.N. and Stevenson, K.J. (1992) Biochem. Cell Biol., 70, 656-663.
3. Jolley, K.A., Rapaport, E., Hough, D.W., Danson, M.J., Woods, W.G., Dyall-Smith, M.L. (1996) J. Bacteriol., 178, 3044-3048.
4. Jolley, K.A., Russell, R.J.M., Hough, D.W. & Danson, M.J. (1997) Eur. J. Biochem., 248, 362- 368.
