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FEBS Letters 580 (2006) 1198–1204

Glyceraldehyde dehydrogenases from the thermoacidophilic torridus and acidophilum, key enzymes of the non-phosphorylative Entner–Doudoroff pathway, constitute a novel enzyme family within the superfamily

Matthias Reher, Peter Scho¨nheit* Institut fu¨r Allgemeine Mikrobiologie, Christian-Albrechts-Universita¨t Kiel, Am Botanischen Garten 1-9, D-24118 Kiel, Germany

Received 9 November 2005; revised 21 December 2005; accepted 11 January 2006

Available online 19 January 2006

Edited by Judit Ova´di

drogenase, subsequent dehydration via gluconate dehydratase Abstract Cells of , grown on glucose, con- tained all enzyme activities of a non-phosphorylative Entner– yields 2-keto-3-deoxygluconate, which is cleaved to pyruvate Doudoroff pathway, including glucose dehydrogenase, gluconate and glyceraldehyde. Glyceraldehyde is then oxidized to glycer- + dehydratase, 2-keto-3-deoxygluconate aldolase, glyceraldehyde ate via a NADP reducing glyceraldehyde dehydrogenase dehydrogenase (GADH), glycerate kinase (2-phosphoglycerate (GADH). A specific kinase phosphorylates glycerate to 2- forming), enolase and pyruvate kinase. GADH was purified to phosphoglycerate, which is then converted to phosphoenoly- homogeneity. The 115-kDa homodimeric protein catalyzed the pyruvate by enolase and further to pyruvate by pyruvate + oxidation of glyceraldehyde with NADP at highest catalytic kinase (Fig. 1) [6,7]. Based on genome data, a non-phosphor- + efficiency. NAD was not used. By MALDI-TOF analysis, open ylative ED pathway was also postulated for P. torridus [2]. reading frame (ORF) Pto0332 was identified in the genome of P. However, with exception of glucose dehydrogenase [8], no en- torridus as the encoding , designated gadh, and the recombi- zyme activities of that pathways had yet been demonstrated. nant GADH was characterized. In ORF Ta0809 represents a gadh homolog with highest sequence Here we report that extracts of glucose-grown P. torridus con- identity; the gene was expressed and the recombinant protein tained all enzyme activities of a non-phosphorylative ED path- + was characterized as functional GADH with properties very sim- way, as reported for Thermoplasma, including a NADP ilar to the P. torridus enzyme. Sequence comparison and phylo- reducing GADH. Further we describe the purification and genetic analysis define both GADHs as members of novel characterization of GADH from P. torridus and the identifica- enzyme family within the aldehyde dehydrogenase superfamily. tion of its encoding gene, designated gadh, by MALDI-TOF 2006 Federation of European Biochemical Societies. Published analysis. In T. acidophilum the gadh homolog was identified, by Elsevier B.V. All rights reserved. expressed and the recombinant protein characterized as GADH with properties very similar as the Picrophilus enzyme. Keywords: Non-phosphorylative Entner–Doudoroff pathway; Sequence comparison, phylogenetic analysis and catalytic Glyceraldehyde dehydrogenase; Aldehyde dehydrogenase superfamily; Picrophilus torridus; Thermoplasma acidophilum function define both GADHs as members of a novel enzyme family within the aldehyde dehydrogenase (ALDH) superfamily.

2. Materials and methods 1. Introduction P. torridus was grown aerobically at 60 C in either 1 l- Erlenmeyer The thermoacidophilic euryarchaeota Thermoplasma acido- flasks (shaken at 150 rpm) or in a 8-l fermentor (FairmenTec, Ger- philum and Picrophilus torridu