Substrate. In 2007, the succinate-CoA ligase in the hyperthermophilic archaeon Thermococcus kodakaraensis, which structurally resembles the acetate-CoA ligase from Pyrococcus furiosus, was described (21). This enzyme exhibits a subunit domain distribution which is distinct in the heterodimer/ tetramer structure typical for SucCD enzymes. This enzyme showed an extended substrate variety and was also active with isovalerate, 3-methyl thiopropionate, glutarate, adipate, and butyrate. Nevertheless, for succinate-CoA ligases using a classical domain structure, relevant investigations are still missing. In 1957, the formation of itaconyl-CoA from itaconate, a structural analogue to succinate, in mammalian liver mitochondria catalyzed by SucCD was re-Received 12 September 2013 Accepted 10 October 2013 Published ahead of print 18 October 2013 Address correspondence to Alexander Steinb hel, [email protected]. Supplemental material for this article could be identified at http://dx.doi.org/10.1128 /AEM.03075-13. Copyright 2014, American Society for Microbiology. All Rights Reserved. doi:ten.1128/AEM.03075-aem.asm.orgApplied and Environmental Microbiologyp. 166 January 2014 Volume 80 NumberCharacterization of Succinate-CoA LigasesFIG 1 Succinate and analogous compounds investigated as possible substrates for SucCD enzymes.ported (22). Later, this reaction was proved for the SucCD from Micrococcus sp. and Pseudomonas fluorescens (224). Later, other investigators showed the participation with the SucCD of Advenella mimigardefordensis DPN7T in the degradation pathway of 3,3=-dithiodipropionic acid (DTDP), a precursor for the production of polythioesters in bacteria (258). In this strain, DTDP is metabolized by way of the intermediate product 3-sulfinopropionate (3SP) (291). This xenobiotic structural analogue to succinate carries a sulfino group rather from the carboxyl group in succinate, and it’s converted to 3SP-CoA in vivo (26). The authors also showed that the mutant strain A. mimigardefordensis DPN7T sucCD was no longer capable to grow on DTDP and 3SP. Additionally, Sch mann et al. demonstrated the conversion of itaconate to itaconyl-CoA by SucCD from A. mimigardefordensis DPN7T (SucCDAm) in vitro (26). As well as that, the authors observed the formation of 3SP-CoA by a crude extract on the expression strain Escherichia coli BL21(DE3)/pLysS not harboring genes for SucCDAm (26). These findings suggested that the formation of 3SP-CoA from 3SP will not be a special characteristic with the A. mimigardefordensis DPN7T SucCD, and it raised the question of whether SucCD enzymes in general have an extended substrate variety like other members of enzyme subsubclass 6.Tirapazamine two.Maraviroc 1 and whether or not other SucCD enzymes are also in a position to form itaconyl-CoA and 3SP-CoA.PMID:24118276 Within this study, we purified three homologous SucCD enzymes and characterized these enzymes with regard to their capability to convert unique carbon acid substrates as analogues of succinate to their corresponding CoA-thioesters in vitro. These integrated the SucCD enzyme from Escherichia coli BL21 (SucCDBL21), SucCDAm, and also the SucCD enzyme from Alcanivorax borkumensisSK2 (SucCDAboHis). SucCDBL21 is identical to the E. coli K-12 (ATCC 47076) enzyme (32) and has been of scientific interest for the last 60 years. The enzyme is able to utilize ATP as well as GTP as a cosubstrate (15). Quite a few crystallographic structures proved the locations on the binding domains for the nucleotide and the CoA involved in catalysis (11, 33). A. mimigardefor.