Purification and properties of the lipoate protein ligase of <i>Escherichia coli</i>

Green, D E; Morris, Timothy W.; Green, J; Cronan, John E.; Guest, John R.

doi:10.1042/bj3090853

Cited by 110 publications

(121 citation statements)

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“…S4B), for cocrystallization. Sulfamoyladenosine analogs have been used to generate competitive inhibitors of aminoacyl-tRNA synthetases (23), and biotin sulfamoyladenosine is a competitive inhibitor of E. coli biotin ligase (24), an enzyme structurally and functionally homologous to LplA (25,26). We confirmed that resorufin ligase was able to bind resorufin sulfamoyladenosine and proceeded to crystallize the ligase in the presence of this analog.…”

Section: Structure-based Mutagenesis and Screening For Resorufin Ligasementioning

confidence: 55%

Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

Liu¹,

Nivón²,

Richter

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of the intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies.fluorescence microscopy | enzyme redesign | LplA | PRIME | chemical probe targeting F luorescent proteins are used ubiquitously in imaging, but their dim fluorescence, rapid photobleaching, and large size limit their utility. At ∼27 kDa (∼240 aa), fluorescent proteins can disrupt protein folding and trafficking or impair protein function (1, 2). Chemical fluorophores, in comparison, are typically less than 1 kDa in size, and are brighter and more photostable. These properties allow chemical fluorophores to perform better than fluorescent proteins in advanced imaging modalities such as singlemolecule tracking and superresolution microscopies (3, 4).Site-specific labeling of proteins with chemical fluorophores inside living cells is challenging because these fluorophores are not genetically encodable and therefore must be posttranslationally targeted inside a complex cellular milieu. Existing methods to achieve this targeting either require large fusion tags [such as HaloTag (5), the SNAP tag (6), and the DHFR tag (7)] or have insufficient specificity [such as biarsenical dye targeting (8) and amber codon suppression (9)]. To achieve a labeling specificity comparable to fluorescent proteins, we developed PRIME (PRobe Incorporation Mediated by Enzymes), which uses Escherichia coli lipoic acid ligase to attach small molecules to a 13-aa peptide tag (Fig. 1A) (10). To make PRIME more useful for cellular protein imaging, we sought to engineer the system for the targeting of bright chemical fluorophores. The challenge, though, is that lipoic acid ligase (LplA) has a small and fully enclosed substrate-binding pocket that even with extensive structure-guided mutagenesis has not until this point been able to accommodate large chemical structures. ResultsSynthesis of Resorufin Derivatives for PRIME. We considered fluorophores for PRIME based on the steric constraints of LplA. Far-red emitters such as Cy5 and Atto 647N, although photophysically desirable, are so bulky that binding inside LplA would require...

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Section: Structure-based Mutagenesis and Screening For Resorufin Ligasementioning

confidence: 55%

Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

Liu¹,

Nivón²,

Richter

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…LplA activates and transfers not only naturally occurring R-(ϩ)-lipoate but also S-(Ϫ)-lipoate, lipoate analogues, and octanoate to the apoproteins, although S-lipoate and octanoate are used at a 44 and 16% rate of R-lipoate, respectively (5,6). LplA has a molecular mass of 37,795 Da, consisting of 337 amino acids excluding the initiating methionine residue, which is cleaved off during the biosynthesis (5).…”

Section: Reactionmentioning

confidence: 99%

Crystal Structure of Lipoate-Protein Ligase A from Escherichia coli

Fujiwara

Toma

Okamura‐Ikeda

et al. 2005

Journal of Biological Chemistry

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Lipoate-protein ligase A (LplA) catalyzes the formation of lipoyl-AMP from lipoate and ATP and then transfers the lipoyl moiety to a specific lysine residue on the acyltransferase subunit of ␣-ketoacid dehydrogenase complexes and on H-protein of the glycine cleavage system. The lypoyllysine arm plays a pivotal role in the complexes by shuttling the reaction intermediate and reducing equivalents between the active sites of the components of the complexes. We have determined the X-ray crystal structures of Escherichia coli LplA alone and in a complex with lipoic acid at 2.4 and 2.9 Å resolution, respectively. The structure of LplA consists of a large N-terminal domain and a small C-terminal domain. The structure identifies the substrate binding pocket at the interface between the two domains. Lipoic acid is bound in a hydrophobic cavity in the N-terminal domain through hydrophobic interactions and a weak hydrogen bond between carboxyl group of lipoic acid and the Ser-72 or Arg-140 residue of LplA. No large conformational change was observed in the main chain structure upon the binding of lipoic acid.Lipoic acid is a prosthetic group of acyltransferase (E2) subunit of the pyruvate, ␣-ketoglutarate, and branched-chain ␣-ketoacid dehydrogenase complexes and of H-protein of the glycine cleavage system (1-4). It attaches to a specific lysine residue on the proteins via an amide linkage between the ⑀-amino group of the lysine residue and the carboxyl group of lipoic acid. In the reaction sequence of the complexes, the lypoyllysine arm shuttles the reaction intermediates and reducing equivalents between the active sites of the components of the complexes.The attachment of lipoic acid to the proteins occurs by two-step reactions in which a lipoyl-AMP intermediate is formed from lipoic acid and ATP, and pyrophosphate is released in the initial activation reaction (Reaction 1).The lipoyl moiety of the intermediate is then transferred to apoproteins in the second transfer reaction, yielding the lipoylated protein and AMP (Reaction 2).Lipoyl-AMP ϩ apoprotein 3 lipoylated protein ϩ AMP (5, 6). LplA has a molecular mass of 37,795 Da, consisting of 337 amino acids excluding the initiating methionine residue, which is cleaved off during the biosynthesis (5). Strains with lplA null mutations have severe defects in the incorporation of exogenously supplied lipoic acid and lipoic acid analogues into apoproteins (5, 7). In E. coli, there is another enzyme, LipB, responsible for the covalent attachment of lipoic acid. LipB transfers lipoic acid/octanoic acid endogenously synthesized on the acyl carrier protein by the function of LipA to the lipoate-dependent enzymes (7-9). LipB consists of 213 amino acids, whose amino acid sequence shares only 12.7% identity with that of LplA (Fig. 1). On the other hand, the amino acid sequence of LplA shows 31 and 35% identity with those of human and bovine lipoyltransferase, the mammalian LplA homologues, respectively (Fig. 1). However, the mammalian lipoyltransferases have no ability to activate l...

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“…In E. coli, lipoyl groups can be incorporated by at least two routes (Figure 1). Scavenged lipoic acid can be utilized by ATP dependent activation and attachment via an amide bond to the lysine residue of the LCD in a reaction catalyzed by the LplA [2]. Alternatively, de novo lipoyl cofactor biosynthesis proceeds initially by octanoylation of the LCD lysine residue [3].…”

Section: Introductionmentioning

confidence: 99%

Structures of lipoyl synthase reveal a compact active site for controlling sequential sulfur insertion reactions

Harmer

Hiscox

Dinis

et al. 2014

Biochemical Journal

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Summary Statement: The biosynthesis of lipoyl cofactors requires two lipoyl synthase mediated sulfur insertions. We report the crystal structures of a lipoyl synthase complexed with S-adenosylhomocysteine or 5'-methylthioadenosine. Models based on these structures identify likely substrate binding sites.Keywords: radical SAM, cofactors, crystal structure, enzyme catalysis, sulfur Abbreviations used: LipA, lipoyl synthase; BioB, biotin synthase; SAM, Sadenosylmethionine; LCD, lipoyl carrier domain; MTA, 5'-methylthioadenosine; RS, radical SAM; ACP, acyl carrier protein; SsLipA, Sulfolobus solfataricus LipA; TeLipA2 Thermosynechococcus elongatus LipA2; Ec, Escherichia coli; 5'-dA, 5'-deoxyadenosine. 2 ABSTRACTLipoyl cofactors are essential for living organisms and are produced by the insertion of two sulfur atoms into the relatively unreactive C-H bonds of an octanoyl substrate. This reaction requires lipoyl synthase, a member of the radical SAM enzyme superfamily. Herein we present crystal structures of lipoyl synthase with two [4Fe-4S] clusters bound at opposite ends of the TIM barrel, the usual fold of the radical SAM superfamily. The cluster required for reductive SAM cleavage conserves the features of the radical SAM superfamily, but the auxiliary cluster is bound by a CX 4 CX 5 C motif unique to lipoyl synthase. The fourth ligand to the auxiliary cluster is an extremely unusual serine residue. Site directed mutants show this conserved serine ligand is essential for the sulfur insertion steps. One crystallized LipA complex contains MTA, a breakdown product of SAM, bound in the likely SAM binding site. Modelling has identified an 18 Å deep channel, well-proportioned to accommodate an octanoyl substrate. These results suggest the auxiliary cluster is the likely sulfur donor, but access to a sulfide ion for the second sulfur insertion reaction requires the loss of an iron atom from the auxiliary cluster, which the serine ligand may enabled.3

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Purification and properties of the lipoate protein ligase of Escherichia coli

Cited by 110 publications

References 46 publications

Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

Crystal Structure of Lipoate-Protein Ligase A from Escherichia coli

Structures of lipoyl synthase reveal a compact active site for controlling sequential sulfur insertion reactions

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