Bout extended ranges concerning the acid substrates for the enzymes happen to be published. The greatest flexibility was shown for longchainSfatty acid CoA ligases (EC six.two.1.three) (16), but acetateCoA ligases (17, 18), a propionateCoA ligase (19), and a butyrateCoA ligase (20) had been also shown to react with extra than 1 carbon acid substrate. In 2007, the succinateCoA ligase from the hyperthermophilic archaeon Thermococcus kodakaraensis, which structurally resembles the acetateCoA ligase from Pyrococcus furiosus, was described (21). This enzyme exhibits a subunit domain distribution which can be distinct in the heterodimer/ tetramer structure standard for SucCD enzymes. This enzyme showed an extended substrate variety and was also active with isovalerate, 3methyl thiopropionate, glutarate, adipate, and butyrate. Nonetheless, for succinateCoA ligases with a classical domain structure, relevant investigations are nevertheless missing. In 1957, the formation of itaconylCoA from itaconate, a structural analogue to succinate, in mammalian liver mitochondria catalyzed by SucCD was reReceived 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 might be found at http://dx.doi.org/10.1128 /AEM.0307513. Copyright 2014, American Society for Microbiology. All Rights Reserved. doi:ten.1128/AEM.03075aem.asm.orgApplied and Environmental Microbiologyp. 166 January 2014 Volume 80 NumberCharacterization of SuccinateCoA LigasesFIG 1 Succinate and analogous compounds investigated as prospective 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 in 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 means of the intermediate item 3sulfinopropionate (3SP) (291). This xenobiotic structural analogue to succinate carries a sulfino group instead with the carboxyl group in succinate, and it is actually converted to 3SPCoA in vivo (26). The authors also showed that the mutant strain A.Methyl 4-chloro-3-methylpicolinate site mimigardefordensis DPN7T sucCD was no longer capable to develop on DTDP and 3SP.Price of XPhos Pd G2 Additionally, Sch mann et al.PMID:23329319 demonstrated the conversion of itaconate to itaconylCoA by SucCD from A. mimigardefordensis DPN7T (SucCDAm) in vitro (26). In addition to that, the authors observed the formation of 3SPCoA by a crude extract from the expression strain Escherichia coli BL21(DE3)/pLysS not harboring genes for SucCDAm (26). These findings suggested that the formation of 3SPCoA from 3SP is just not a exclusive characteristic with the A. mimigardefordensis DPN7T SucCD, and it raised the question of whether or not SucCD enzymes in general have an extended substrate variety like other members of enzyme subsubclass 6.two.1 and irrespective of whether other SucCD enzymes are also in a position to type itaconylCoA and 3SPCoA. In this study, we purified three homologous SucCD enzymes and characterized these enzymes with regard to their capability to convert distinctive carbon acid substrates as analogues of succinate to their corresponding CoAthioesters in vitro. These included the SucCD enzyme from Escherichia coli BL21 (SucCDBL21), SucCDAm, and the SucCD enzyme from Alcanivorax borkumensisSK2 (SucCDAboHis). SucCDBL21 is identical towards the E. coli K12 (AT.