Gene and primary structures of dyelinked Emphasi

Authors: Ryushi Kawakami Haruhiko Sakuraba Toshihisa Ohshima

Publish Date: 2003/12/12

Volume: 8, Issue: 2, Pages: 99-108

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Abstract

Dyelinked lproline dehydrogenase catalyzes the oxidation of lproline in the presence of artificial electron acceptors such as 2 6dichloroindophenol and ferricyanide The enzyme from the hyperthermophilic archaeon Thermococcus profundus was purified and characterized for the first time in archaea by Sakuraba et al in 2001 In this study cloning and sequencing analyses of the gene encoding the enzyme and functional analysis of the subunits were performed The gene formed an operon that consisted of four genes pdhA pdhB pdhF and pdhX which are tandemly arranged in the order of pdhAFXB SDSPAGE analysis of the purified recombinant enzyme showed four different bands corresponding to α 54 kDa β 43 kDa γ 19 kDa and δ 8 kDa subunits encoded by pdhA pdhB pdhF and pdhX respectively and the molecular ratio of these subunits was determined to be equal This indicates that the enzyme consists of a heterotetrameric αβγδ structure Functional analysis of each subunit revealed that the β subunit catalyzed the dyelinked lproline dehydrogenase reaction by itself and that unexpectedly the α subunit exhibited dyelinked NADH dehydrogenase activity This is the first example showing the existence of a bifunctional dyelinked lproline/NADH dehydrogenase complex On the basis of genome analysis similar gene clusters were observed in the genomes of Pyrococcus horikoshii Pyrococcus abyssi Pyrococcus furiosus and Archaeoglobus fulgidus These results indicate that the dyelinked lproline dehydrogenase is a novel type of heterotetrameric amino acid dehydrogenase that might be widely distributed in the hyperthermophilic archaeal strain

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