Allels between LipA and biotin synthase (BioB), the enzyme discussed above

Allels between LipA and biotin synthase (BioB), the enzyme discussed above that catalyzes the final step in the biotin biosynthesis (Fig. 4). LipA like BioB makes two C-S bonds and also removes two unactivated hydrogen atoms. The similarity in chemistry between the biosynthesis of the dithiolane ring of lipoate and the thiophane ring of biotin strongly suggests functional parallels in the mechanisms of the enzymes that produce these compounds. Indeed, the amino acid sequences of the E. coli LipA and BioB proteins show marked similarities; 40 sequence similarity and 17 sequence identity (217). As discussed above BioB has both a [4Fe-4S] cluster and a [2Fe-2S] cluster. The canonical iron-sulfur cluster binding motif CXXXCXXC is also found in the LipA sequence leading to early predictions that it is an iron-sulfur protein (217, 236). The LipA protein has been overexpressed in E. coli and purified from both soluble lysates and insoluble aggregates that were subsequently refolded and reconstituted with ferrous iron and sulfide (217, 237, 238). The purified dimeric protein (237) has a dark reddish-brown color and displays a band at 420 nm in its light absorption spectrum, characteristic of a sulfide to iron charge transfer. Resonance Raman, electronic absorbance and M purchase Velpatasvir sbauer spectroscopic results were consistent with the presence of an iron-sulfur cluster in LipA. It was suggested that LipA contains [2Fe-2S] clusters that during reduction are converted into [4Fe-4S] clusters (237, 238). The Fe-S cluster of LipA was first suggested to be a [4Fe-4S] cluster bridging the two subunits (237). However, in a different report it was suggested that the limited amount of Fe and S atoms and the presence of [2Fe-2S] clusters in the previous preparation of LipA were a direct consequence of aerobic isolation. It was reported that under strictly anaerobic conditions LipA could bind one [4Fe-4S] cluster per subunit (239). Recently it was reported that LipA contains two distinct [4Fe-4S] clusters per polypeptide (203). Thus, the types ofAuthor Manuscript Author Manuscript Author Manuscript Author ManuscriptEcoSal Plus. Author manuscript; available in PMC 2015 January 06.CronanPagedisagreements seen in the BioB Monocrotaline site literature are also apparent for LipA showing the obvious difficulties of working with this family of proteins Direct involvement of LipA in the sulfur insertion reaction of lipoic acid biosynthesis was difficult to establish due to the lack of an in vitro assay. Much of this difficulty was due to the assumption that free octanoic acid was the sulfur acceptor. The first indication that this was not the case was the demonstration by Jordan and Cronan (218) of the LipB transferase activity. Miller and coworkers (240) were the first to report synthesis of lipoic acid in vitro. This was based on the discovery of LplA and LipB which led to development of a defined in vitro lipoic acid synthesis system and an assay that was much more sensitive and quantitative than prior assays (240). Lipoic acid synthesis was assayed indirectly using (i) the apo form of pyruvate dehydrogenase complex (apo-PDH) as a lipoyl-accepting protein, (ii) purified LipA, and either (iii) purified LplA, ATP, octanoic acid as a substrate (for lipoic acid synthesis) or iv) LipB and octanoyl-ACP as a substrate. Activation of apo-PDH upon lipoylation was monitored spectrophotometrically via reduction of an NAD+ analogue. Within a finite range, the rate of reduced pyridine dinucleotide for.Allels between LipA and biotin synthase (BioB), the enzyme discussed above that catalyzes the final step in the biotin biosynthesis (Fig. 4). LipA like BioB makes two C-S bonds and also removes two unactivated hydrogen atoms. The similarity in chemistry between the biosynthesis of the dithiolane ring of lipoate and the thiophane ring of biotin strongly suggests functional parallels in the mechanisms of the enzymes that produce these compounds. Indeed, the amino acid sequences of the E. coli LipA and BioB proteins show marked similarities; 40 sequence similarity and 17 sequence identity (217). As discussed above BioB has both a [4Fe-4S] cluster and a [2Fe-2S] cluster. The canonical iron-sulfur cluster binding motif CXXXCXXC is also found in the LipA sequence leading to early predictions that it is an iron-sulfur protein (217, 236). The LipA protein has been overexpressed in E. coli and purified from both soluble lysates and insoluble aggregates that were subsequently refolded and reconstituted with ferrous iron and sulfide (217, 237, 238). The purified dimeric protein (237) has a dark reddish-brown color and displays a band at 420 nm in its light absorption spectrum, characteristic of a sulfide to iron charge transfer. Resonance Raman, electronic absorbance and M sbauer spectroscopic results were consistent with the presence of an iron-sulfur cluster in LipA. It was suggested that LipA contains [2Fe-2S] clusters that during reduction are converted into [4Fe-4S] clusters (237, 238). The Fe-S cluster of LipA was first suggested to be a [4Fe-4S] cluster bridging the two subunits (237). However, in a different report it was suggested that the limited amount of Fe and S atoms and the presence of [2Fe-2S] clusters in the previous preparation of LipA were a direct consequence of aerobic isolation. It was reported that under strictly anaerobic conditions LipA could bind one [4Fe-4S] cluster per subunit (239). Recently it was reported that LipA contains two distinct [4Fe-4S] clusters per polypeptide (203). Thus, the types ofAuthor Manuscript Author Manuscript Author Manuscript Author ManuscriptEcoSal Plus. Author manuscript; available in PMC 2015 January 06.CronanPagedisagreements seen in the BioB literature are also apparent for LipA showing the obvious difficulties of working with this family of proteins Direct involvement of LipA in the sulfur insertion reaction of lipoic acid biosynthesis was difficult to establish due to the lack of an in vitro assay. Much of this difficulty was due to the assumption that free octanoic acid was the sulfur acceptor. The first indication that this was not the case was the demonstration by Jordan and Cronan (218) of the LipB transferase activity. Miller and coworkers (240) were the first to report synthesis of lipoic acid in vitro. This was based on the discovery of LplA and LipB which led to development of a defined in vitro lipoic acid synthesis system and an assay that was much more sensitive and quantitative than prior assays (240). Lipoic acid synthesis was assayed indirectly using (i) the apo form of pyruvate dehydrogenase complex (apo-PDH) as a lipoyl-accepting protein, (ii) purified LipA, and either (iii) purified LplA, ATP, octanoic acid as a substrate (for lipoic acid synthesis) or iv) LipB and octanoyl-ACP as a substrate. Activation of apo-PDH upon lipoylation was monitored spectrophotometrically via reduction of an NAD+ analogue. Within a finite range, the rate of reduced pyridine dinucleotide for.

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