Sulfatases catalyze the cleavage of a variety of cellular sulfate esters via a novel mechanism that requires the action of a protein-derived formylglycine cofactor. Formation of the cofactor is catalyzed by an accessory protein and involves the two-electron oxidation of a specific cysteinyl or seryl residue on the relevant sulfatase. Although some sulfatases undergo maturation via mechanisms in which oxygen serves as an electron acceptor, AtsB, the maturase from Klebsiella pneumoniae, catalyzes the oxidation of Ser72 on AtsA, its cognate sulfatase, via an oxygen-independent mechanism. Moreover, it does not make use of pyridine or flavin nucleotide cofactors as direct electron acceptors. In fact, AtsB has been shown to be a member of the radical S-adenosylmethionine superfamily of proteins, suggesting that it catalyzes this oxidation via an intermediate 5′-deoxyadenosyl 5′-radical that is generated by a reductive cleavage of S-adenosyl-L-methionine. In contrast to AtsA, very little in vitro characterization of AtsB has been conducted. Herein we show that coexpression of the K. pneumoniae atsB gene with a plasmid that encodes genes that are known to be involved in ironsulfur cluster biosynthesis yields soluble protein that can be characterized in vitro. The as-isolated protein contained 8.7 ± 0.4 irons and 12.2 ± 2.6 sulfides per polypeptide, which existed almost entirely in the [4Fe-4S] 2+ configuration, as determined by Mössbauer spectroscopy, suggesting that it contained at least two of these clusters per polypeptide. Reconstitution of the as-isolated protein with additional iron and sulfide indicated the presence of 12.3 ± 0.2 irons and 9.9 ± 0.4 sulfides per polypeptide. Subsequent characterization of the reconstituted protein by Mössbauer spectroscopy indicated the presence of only [4Fe-4S] clusters, suggesting that reconstituted AtsB contains three per polypeptide. Consistent with this stoichiometry, an as-isolated AtsB triple variant containing Cys → Ala substitutions at each of the cysteines in its CX 3 CX 2 C radical SAM motif contained 7.3 ± 0.1 irons and 7.2 ± 0.2 sulfides per polypeptide while the reconstituted triple variant contained 7.7 ± 0.1 irons and 8.4 ± 0.4 sulfides per polypeptide, indicating that it was unable to incorporate an additional cluster. UV-visible and Mössbauer spectra of both samples indicated the presence of only clusters. AtsB was capable of catalyzing multiple turnovers and exhibited a V max /[E T ] of ~0.36 min −1 for an 18-amino acid peptide substrate using dithionite to supply the requisite electron and a value of ~0.039 min −1 for the same substrate using the physiologically relevant flavodoxin reducing system. Simultaneous quantification of formylglycine and 5′-deoxyadenosine as a function of time indicates an approximate 1:1 stoichiometry. Use of a peptide substrate in which the target serine is † This work was supported by NIH Grant GM-63847 (S.J.B.), the Dreyfus Foundation (Teacher Scholar Award to C.K.), and the Beckman Foundation (Young Investigator Award to...
Lipoyl synthase (LS) is a member of a recently established class of metalloenzymes that use S-adenosyl-l-methionine (SAM) as the precursor to a high-energy 5'-deoxyadenosyl 5'-radical (5'-dA(*)). In the LS reaction, the 5'-dA(*) is hypothesized to abstract hydrogen atoms from C-6 and C-8 of protein-bound octanoic acid with subsequent sulfur insertion, generating the lipoyl cofactor. Consistent with this premise, 2 equiv of SAM is required to synthesize 1 equiv of the lipoyl cofactor, and deuterium transfer from octanoyl-d(15) H-protein of the glycine cleavage system-one of the substrates for LS-has been reported [Cicchillo, R. M., Iwig, D. F., Jones, A. D., Nesbitt, N. M., Baleanu-Gogonea, C., Souder, M. G., Tu, L., and Booker, S. J. (2004) Biochemistry 43, 6378-6386]. However, the exact identity of the sulfur donor remains unknown. We report herein that LS from Escherichia coli can accommodate two [4Fe-4S] clusters per polypeptide and that this form of the enzyme is relevant to turnover. One cluster is ligated by the cysteine amino acids in the C-X(3)-C-X(2)-C motif that is common to all radical SAM enzymes, while the other is ligated by the cysteine amino acids residing in a C-X(4)-C-X(5)-C motif, which is conserved only in lipoyl synthases. When expressed in the presence of a plasmid that harbors an Azotobacter vinelandii isc operon, which is involved in Fe/S cluster biosynthesis, the as-isolated wild-type enzyme contained 6.9 +/- 0.5 irons and 6.4 +/- 0.9 sulfides per polypeptide and catalyzed formation of 0.60 equiv of 5'-deoxyadenosine (5'-dA) and 0.27 equiv of lipoylated H-protein per polypeptide. The C68A-C73A-C79A triple variant, expressed and isolated under identical conditions, contained 3.0 +/- 0.1 irons and 3.6 +/- 0.4 sulfides per polypeptide, while the C94A-C98A-C101A triple variant contained 4.2 +/- 0.1 irons and 4.7 +/- 0.8 sulfides per polypeptide. Neither of these variant proteins catalyzed formation of 5'-dA or the lipoyl group. Mössbauer spectroscopy of the as-isolated wild-type protein and the two triple variants indicates that greater than 90% of all associated iron is in the configuration [4Fe-4S](2+). When wild-type LS was reconstituted with (57)Fe and sodium sulfide, it harbored considerably more iron (13.8 +/- 0.6) and sulfide (13.1 +/- 0.2) per polypeptide and catalyzed formation of 0.96 equiv of 5'-dA and 0.36 equiv of the lipoyl group. Mössbauer spectroscopy of this protein revealed that only approximately 67% +/- 6% of the iron is in the form of [4Fe-4S](2+) clusters, amounting to 9.2 +/- 0.4 irons and 8.8 +/- 0.1 sulfides or 2 [4Fe-4S](2+) clusters per polypeptide, with the remainder of the iron occurring as adventitiously bound species. Although the Mössbauer parameters of the clusters associated with each of the variants are similar, EPR spectra of the reduced forms of the cluster show small differences in spin concentration and g-values, consistent with each of these clusters as distinct species residing in each of the two cysteine-containing motifs.
Lipoyl synthase (LipA) catalyzes the insertion of two sulfur atoms at the unactivated C6 and C8 positions of a protein-bound octanoyl chain to produce the lipoyl cofactor. To activate its substrate for sulfur insertion, LipA uses a [4Fe-4S] cluster and S-adenosylmethionine (AdoMet) radical chemistry; the remainder of the reaction mechanism, especially the source of the sulfur, has been less clear. One controversial proposal involves the removal of sulfur from a second (auxiliary) [4Fe-4S] cluster on the enzyme, resulting in destruction of the cluster during each round of catalysis. Here, we present two high-resolution crystal structures of LipA from Mycobacterium tuberculosis: one in its resting state and one at an intermediate state during turnover. In the resting state, an auxiliary [4Fe-4S] cluster has an unusual serine ligation to one of the irons. After reaction with an octanoyllysine-containing 8-mer peptide substrate and 1 eq AdoMet, conditions that allow for the first sulfur insertion but not the second insertion, the serine ligand dissociates from the cluster, the iron ion is lost, and a sulfur atom that is still part of the cluster becomes covalently attached to C6 of the octanoyl substrate. This intermediate structure provides a clear picture of iron-sulfur cluster destruction in action, supporting the role of the auxiliary cluster as the sulfur source in the LipA reaction and describing a radical strategy for sulfur incorporation into completely unactivated substrates.iron-sulfur cluster | radical SAM enzyme | lipoic acid T he functionalization of aliphatic carbon centers is widely regarded as one of the most kinetically challenging reactions in nature, a striking example of which is found in the biosynthesis of the lipoyl cofactor, famous for its central role as the "swinging arm" of the pyruvate dehydrogenase enzyme complex. Lipoyl synthase (LipA) generates the lipoyl cofactor by insertion of two sulfur atoms at C6 and C8 of a protein-bound n-octanoyl chain, sites distal from the nearest functionality (1-4). LipA and the closely related biotin synthase (BioB) (Scheme 1) are founding members of the ever-expanding S-adenosyl-L-methionine (AdoMet) radical enzyme superfamily that uses a [4Fe-4S] cluster to reductively cleave the C5′-S bond of AdoMet, generating methionine and a 5′-deoxyadenosyl radical (5′-dA•), a powerful oxidant (5, 6). LipA requires 2 eq AdoMet-one per sulfur insertion-and two sulfur atoms to produce 1 eq lipoyl product through radical-based chemistry (4). One of the most controversial aspects of the LipA and BioB reactions is the source of the sulfur. AdoMet radical enzymes that catalyze sulfur insertion always seem to have an additional iron-sulfur (Fe/S) cluster: a [2Fe-2S] cluster in BioB (7), a [4Fe-4S] cluster in LipA (8), and a [4Fe-4S] cluster in the methylthiotransferases RimO and MiaB (9, 10). These auxiliary clusters in LipA and BioB have been proposed to be cannibalized during turnover to supply the inserted sulfur atom(s) (8, 11-13), a proposal that has not enjoyed univ...
RimO, encoded by the yliG gene in Escherichia coli, has been recently identified in vivo as the enzyme responsible for the attachment of a methylthio group on the β-carbon of Asp88 of the small ribosomal protein S12 [Anton, B. P., Saleh, L., Benner, J. S., Raleigh, E. A., Kasif, S., and Roberts, R. J. (2008) Proc. Natl. Acad. Sci. USA, 105, 1826USA, 105, -1831. To date, it is the only enzyme known to catalyze methylthiolation of a protein substrate; the four other naturally occurring methylthio modifications have been observed on tRNA. All members of the methylthiotransferase (MTTase) family, to which RimO belongs, have been shown to contain the canonical CxxxCxxC motif in their primary structures that is typical of the radical S-adenosylmethionine (SAM) family of proteins. MiaB, the only characterized MTTase, and the enzyme experimentally shown to be responsible for methylthiolation of N 6 -isopentenyladenosine of tRNA in E. coli and Thermotoga maritima, has been demonstrated to harbor two distinct clusters. Herein, we report in vitro biochemical, and spectroscopic characterization of RimO. We show by analytical and spectroscopic methods that RimO, heterologously overproduced in E. coli in the presence of iron-sulfur cluster biosynthesis proteins from Azotobacter vinelandii, contains one [4Fe-4S] 2+ cluster. Reconstitution of this form of RimO (RimO rcn ) with 57 Fe and sodium sulfide results in a protein that contains two [4Fe-4S] 2+ clusters, similar to MiaB. We also show by mass spectrometry that RimO rcn catalyzes the attachment of a methylthio group to a peptide substrate analog that mimics the loop structure bearing aspartyl 88 of the S12 ribosomal protein from E. coli. Kinetic analysis of this reaction shows that the activity of RimO rcn in the presence of the substrate analog does not support a complete turnover. We discuss the possible requirement for an assembled ribosome for fully active RimO in vitro. Our findings are † This work was supported by NIH Grant GM-63847 and NSF Grant MCB-0133826 (S.J.B.), the Dreyfus Foundation (Teacher Scholar Award to C.K.), the Beckman Foundation (Young Investigator Award to C.K.), and New England Biolabs. * To whom correspondence should be addressed. Squire J. Booker, 104 Chemistry Building, The Pennsylvania State University, University Park, PA 16802. Phone: 814-865-8793. Fax: 814-865-2927. Squire@psu.edu. Carsten Krebs, 104 Chemistry Building, The Pennsylvania State University, University Park, PA 16802. Phone: 814-865-6089. Fax: 814-865-2927. ckrebs@psu.edu. Lana Saleh, 240 County Road, Ipswich, MA 01908. Phone: 978-380-7446. Fax: 978-921-1350. Saleh@neb.com. Δ These authors contributed equally to this work 3 The EPR and Mössbauer spectroscopic features of RimO ai are consistent with the presence of a small amount of the SAM-bound form, because the spectral features of the SAM-bound and SAM-free forms overlap heavily. Supporting Information AvailableFigures S1, S2, S3, S4, S5, and S6. This material is available free of charge via the Internet at http...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.