Lipoic acid is a substrate for E2p and E3 in vitro, lipoylated domain is a much better substrate (Graham et al., 1989). Attachment of the lipoyl group to the conserved 11-Deoxojervine site lysine at the tip of the protruding urn gives a dramatic reach to the “business end”. Moreover, the flexible and extended linker regions that connect the lipoyl domain(s) with the catalytic domain contribute increased mobility to the swinging arm since deletion of the linker region in a modified “single lipoyl domain” E2p chain caused an almost total loss of overall activity without substantially affecting the individual enzymatic activities (173). Second E1p and E1o of E. coli (85, 114) and A. vinelandii (174) can only transfer acyl groups to their cognate E2 protein thereby providing an accurate substrate channeling mechanism such that the reductive acylation only occurs on the lipoyl group covalently attached to the appropriate E2 subunit. Third, although the attached lipoate was once thought to be freely rotating (175, 176), recent structural data showed that the lipoyl-lysine urn of the domain became less flexible after lipoylation of the lysine residue (177). The restricted motion of the lipoyl group would facilitate the effective E1 and E2 interaction by presenting the lipoyl group in the preferred orientation to the buy Olumacostat glasaretil active sites of E1 and thereby enhance catalysis. This is in agreement with the recent crystal structure of the E1 component of the BCDH complex from P. putida (178). According to this structure, the active site where thiamine diphosphate binds is at the bottom of a long funnel-shaped tunnel, which suggests that the lipoyl group attached to the lipoyl domain must be fully extended and accurately positioned in order to reach the thiamine diphosphate cofactor. Amino acid side chains responsible for this specific positioning have been mapped to two residues that flank the lipoyl-lysine (179). Finally, the prominent surface loop connecting trands 1 and 2 (which lie close in space to the lipoyllysine) is another major determinant of the interactions of the lipoyl domain with E1 (180). Deletion of this loop results in a partially folded domain and almost completely abolishes lipoylation and reductive acylation indicating that the loop is involved in maintaining the structural integrity of the domain, post-translational modification and catalytic function (177). It is believed that the loop structure is important for stabilizing the thioester bond of the acyl-lipoyl intermediate (177, 181). Subgenes that encode the lipoyl domains from a wide range of bacteria, including E. coli E2p (182) and E2o (183), Bacillus stearothermophilus E2p (184), human E2p (185), Azotobacter vinelandii E2p (166) and E2o (186), and Neisseria meningitidis E2p (187) have been overexpressed in E. coli and sufficient recombinant protein has been obtained for the domain structures to be determined by multidimensional nuclear magnetic resonance (NMR) spectroscopy. The archetypical structure, that of the single apo lipoyl domain of the E2pAuthor Manuscript Author Manuscript Author Manuscript Author ManuscriptEcoSal Plus. Author manuscript; available in PMC 2015 January 06.CronanPagechain of B. stearothermophilus (188), is composed largely of two four-stranded heets, with the N- and C-terminal residues of the domain close together in space in one sheet and the lysine residue earmarked for lipoylation in an exposed position in a tight type I urn generated by trand 4 and 5 in the other.Lipoic acid is a substrate for E2p and E3 in vitro, lipoylated domain is a much better substrate (Graham et al., 1989). Attachment of the lipoyl group to the conserved lysine at the tip of the protruding urn gives a dramatic reach to the “business end”. Moreover, the flexible and extended linker regions that connect the lipoyl domain(s) with the catalytic domain contribute increased mobility to the swinging arm since deletion of the linker region in a modified “single lipoyl domain” E2p chain caused an almost total loss of overall activity without substantially affecting the individual enzymatic activities (173). Second E1p and E1o of E. coli (85, 114) and A. vinelandii (174) can only transfer acyl groups to their cognate E2 protein thereby providing an accurate substrate channeling mechanism such that the reductive acylation only occurs on the lipoyl group covalently attached to the appropriate E2 subunit. Third, although the attached lipoate was once thought to be freely rotating (175, 176), recent structural data showed that the lipoyl-lysine urn of the domain became less flexible after lipoylation of the lysine residue (177). The restricted motion of the lipoyl group would facilitate the effective E1 and E2 interaction by presenting the lipoyl group in the preferred orientation to the active sites of E1 and thereby enhance catalysis. This is in agreement with the recent crystal structure of the E1 component of the BCDH complex from P. putida (178). According to this structure, the active site where thiamine diphosphate binds is at the bottom of a long funnel-shaped tunnel, which suggests that the lipoyl group attached to the lipoyl domain must be fully extended and accurately positioned in order to reach the thiamine diphosphate cofactor. Amino acid side chains responsible for this specific positioning have been mapped to two residues that flank the lipoyl-lysine (179). Finally, the prominent surface loop connecting trands 1 and 2 (which lie close in space to the lipoyllysine) is another major determinant of the interactions of the lipoyl domain with E1 (180). Deletion of this loop results in a partially folded domain and almost completely abolishes lipoylation and reductive acylation indicating that the loop is involved in maintaining the structural integrity of the domain, post-translational modification and catalytic function (177). It is believed that the loop structure is important for stabilizing the thioester bond of the acyl-lipoyl intermediate (177, 181). Subgenes that encode the lipoyl domains from a wide range of bacteria, including E. coli E2p (182) and E2o (183), Bacillus stearothermophilus E2p (184), human E2p (185), Azotobacter vinelandii E2p (166) and E2o (186), and Neisseria meningitidis E2p (187) have been overexpressed in E. coli and sufficient recombinant protein has been obtained for the domain structures to be determined by multidimensional nuclear magnetic resonance (NMR) spectroscopy. The archetypical structure, that of the single apo lipoyl domain of the E2pAuthor Manuscript Author Manuscript Author Manuscript Author ManuscriptEcoSal Plus. Author manuscript; available in PMC 2015 January 06.CronanPagechain of B. stearothermophilus (188), is composed largely of two four-stranded heets, with the N- and C-terminal residues of the domain close together in space in one sheet and the lysine residue earmarked for lipoylation in an exposed position in a tight type I urn generated by trand 4 and 5 in the other.
