Identification of two [4Fe–4S]-cluster-containing hydro-lyases from Pyrococcus furiosus

Abstract

The hyperthermophilic archaeon Pyrococcus furiosus is a strict anaerobe. It is therefore not expected to use the oxidative tricarboxylic acid (TCA) cycle for energy transduction. Nonetheless, its genome encodes more putative TCA cycle enzymes than the closely related Pyrococcus horikoshii and Pyrococcus abyssi, including an aconitase (PF0201). Furthermore, a two-subunit fumarase (PF1755 and PF1754) is encoded on the Pyr. furiosus genome. In the present study, these three genes were heterologously overexpressed in Escherichia coli to enable characterization of the enzymes. PF1755 and PF1754 were shown to form a [4Fe-4S]-clustercontaining heterodimeric enzyme, able to catalyse the reversible hydratation of fumarate. The aconitase PF0201 also contained an Fe-S cluster, and catalysed the conversion from citrate to isocitrate. The fumarase belongs to the class of two-subunit, [4Fe-4S]-cluster-containing fumarate hydratases exemplified by MmcBC from Pelotomaculum thermopropionicum; the aconitase belongs to the aconitase A family. Aconitase probably plays a role in amino acid synthesis when the organism grows on carbohydrates. However, the function of the seemingly metabolically isolated fumarase in Pyr. furiosus has yet to be established.

Biochemical Similarities and Differences between the Catalytic [4Fe-4S] Cluster Containing Fumarases FumA and FumB from Escherichia coli

“…Therefore meso-tartrate is not a substrate, while D-tartrate is. The same stereospecificity has also been found for the type II fumarases such as E. coli FumC and for the P. furiosus fumarase [5], [7].…”

“…The iron-independent, oxygen-stable FumC belongs to class II [6] and is homologous to eukaryotic fumarases. More recently a third type of fumarase was found in the thermophilic bacterium Pelotomaculum thermopropionicum and hyperthermophilic archaeon Pyrococcus furiosus , which is heterodimeric and also relies on a [4Fe-4S] cluster as a catalytic centre [7], [8].…”

Identification and characterization of a novel fumarase gene by metagenome expression cloning from marine microorganisms

“…The K m value for FumF protein was similar to that of the MmcBC fumarase from Pelotomaculum thermopropionicum strain SI (DSM 13744) [7]. A more complete analysis of the optimal reaction conditions and properties of FumF protein could enable better industrial production of L -malate under higher temperature conditions [25]. Furthermore, elucidating the catalytic mechanism through studies of the 3 D structures of FumF protein by X-ray diffraction could define protein regions that determine catalytic rates.…”

Caracterização estrutural e funcional das isoformas da enzima fumarato hidratase de Trypanosoma cruzi

“…This type of spectra was assigned to [3Fe-4S] + clusters (Flint, Emptage e Guest, 1992). This result differs from previously published data which reported that [4Fe-4S] 2+ clusters are present in the structures of other fumarate hydratases (Flint, Emptage e Guest, 1992;Vankuijk et al, 1996;Shimoyama et al, 2007;Van Vugt-Lussenburg et al, 2009). [4Fe-4S] clusters are described to be crucial to class I fumarases catalytic mechanism where they behave as a Lewis acid that activates the substrates (Flint, Emptage e Guest, 1992).…”