Metabolic coupling of two small-molecule thiols programs the biosynthesis of lincomycin A


Low-molecular-mass thiols in organisms are well known for their redox-relevant role in protection against various endogenous and exogenous stresses1,2,3. In eukaryotes and Gram-negative bacteria, the primary thiol is glutathione (GSH), a cysteinyl-containing tripeptide. In contrast, mycothiol (MSH), a cysteinyl pseudo-disaccharide, is dominant in Gram-positive actinobacteria, including antibiotic-producing actinomycetes and pathogenic mycobacteria. MSH is equivalent to GSH, either as a cofactor or as a substrate, in numerous biochemical processes4, most of which have not been characterized, largely due to the dearth of information concerning MSH-dependent proteins. Actinomycetes are able to produce another thiol, ergothioneine (EGT), a histidine betaine derivative that is widely assimilated by plants and animals for variable physiological activities5. The involvement of EGT in enzymatic reactions, however, lacks any precedent. Here we report that the unprecedented coupling of two bacterial thiols, MSH and EGT, has a constructive role in the biosynthesis of lincomycin A, a sulfur-containing lincosamide (C8 sugar) antibiotic that has been widely used for half a century to treat Gram-positive bacterial infections6,7,8,9. EGT acts as a carrier to template the molecular assembly, and MSH is the sulfur donor for lincomycin maturation after thiol exchange. These thiols function through two unusual S-glycosylations that program lincosamide transfer, activation and modification, providing the first paradigm for EGT-associated biochemical processes and for the poorly understood MSH-dependent biotransformations, a newly described model that is potentially common in the incorporation of sulfur, an element essential for life and ubiquitous in living systems.

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Figure 1: Representative low-molecular-mass thiols, relevant metabolic pathways, and associated lincosamide natural products.
Figure 2: Characterization of LmbE as a pathway-specific Mca protein to process 1, the MSH S-conjugated intermediate, in the biosynthesis of lincomycin A.
Figure 3: Characterization of MSH- and EGT-associated biotransformations.
Figure 4: The EGT (pink) and MSH (blue) programmed biosynthetic pathway of lincomycin A.

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Data deposits

The sequences of the genes lmbE80, lmbE447, lmbE3457, mshAlin and egtDlin are deposited in GenBank with the NCBI accession numbers KJ958528, KJ958529, KJ958530, KJ958531 and KJ958532, respectively.


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This work was supported in part by grants from NSFC (81302674, 31430005, 91213303, 21472231 and 91413101), STCSM (14JC1407700 and 13XD1404500) and MST (2012AA02A706) of China.

Author information




Q.Zhao, M.W. and Q.Zhang performed in vivo investigations. M.W. and D.X. prepared and characterized the chemical compounds. Q.Zhao and W.L. conducted sequence analysis. Q.Zhao and M.W. performed in vitro enzymatic investigations. Q.Zhao, M.W. and W.L. analysed the data and wrote the manuscript. W.L. directed the research.

Corresponding author

Correspondence to Wen Liu.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Constructive role of MSH (orange) and EGT (green) in lincomycin biosynthesis (shown as cartoon models).

Extended Data Figure 2 Genes and/or clusters relevant to lincomycin biosynthesis in S. lincolnensis.

a, Biosynthetic gene cluster of lincomycin A. The mca homologue lmbE and two GTase-encoding genes, lmbV and lmbT, are shown in yellow, blue and green, respectively. The genes responsible for PPL incorporation (lmbC, lmbD and lmbN) are shown in grey. b, The location of mshAlin (shown in purple), which is not clustered with the other genes responsible for MSH biosynthesis, in the genome of S. lincolnensis. The flanking genes lin5590, lin5591, lin5593 and lin5594 share sequence homology with genes encoding the methylase (WP_026151264.1) from S. prunicolor, the DUF899-like protein (WP_004004056.1) from S. viridochromogenes, the YbjN-like protein (WP_020130332.1) from Streptomyces sp. 303MFCol5.2 and the hypothetical protein (WP_004000160.1) from S. viridochromogenes, respectively. c, The biosynthetic gene cluster of EGT. The gene egtDlin is shown in red.

Extended Data Figure 3 The production of MSH S-conjugate 1 and lincomycin A in various S. lincolnensis strains.

The strains include the mutants (i, for ΔlmbE; ii, for ΔlmbE-E80; iii, for ΔlmbE-E447; and iv, for ΔlmbE-E3457) and the wild-type control (v). For HPLC-MS analysis, the ESI m/z [M + H]+ modes are indicated in the dashed rectangle.

Extended Data Figure 4 Analysis of MSH and EGT production in the ΔmshAlin and ΔegtDlin mutant S. lincolnensis strains.

a, Derivatization of MSH and EGT with mBBr to generate the corresponding S-conjugates. b, HPLC-HR-MS analysis of MSH-mBBr (calculated for C27H41N4O14S+ [M + H]+ 677.2340) and EGT-mBBr (calculated for C19H26N5O4S+ [M + H]+ 420.1706) in the wild-type control (i), LL1005 (ii, ΔmshAlin mutant) and LL1010 (iii, ΔegtDlin mutant). c, The HR-ESI-MS spectra of EGT-mBBr (top) and MSH-mBBr (below).

Extended Data Figure 5 Phylogenetic analysis in the DinB-2 superfamily.

a, LmbV (from S. lincolnensis) and CcbV (from S. caelestis), shown in red, with selected DinB-2-like proteins (from various actinomycetes, in which the thiol MSH is dominant) in the phylogenetic tree. The evolutionary distances were computed using the p-distance method. The support for grouping clades i, ii, iii, iv and v (shaded in different colours) is indicated by bootstrap values. The known MSH-maleylpyruvate isomerase Ncgl2918 is shown in blue. b, Typical domain organization of the DinB-2-like proteins. The conserved DinB-2 domain is shown in green. Clade i features the C-terminal MDMPI-C domain. Proteins containing this domain include the MSH-maleylpyruvate isomerases, such as Ncgl2918. Clade ii features the C-terminal FGE-sulfatase domain, which is found in eukaryotic proteins required for post-translational modification to produce sulfatases, which are essential for the degradation and remodelling of sulfate esters. Clade iii features the N-terminal zf-HC2 domain, which contains a putative zinc-finger binding motif and is found in some anti-sigma factor proteins. Clade iv features the C-terminal SCP2 domain involved in binding sterols. Clade v features the C-terminal wyosine_f domain. Some proteins containing this domain appear to be important in wyosine base formation in a subset of phenylalanine-specific tRNAs. ‘Others’ indicate a number of DinB-2-like proteins that possess an unknown domain(s) either at the C terminus or at both the C and N termini of the proteins.

Extended Data Figure 6 Kinetic analysis of CcbV-catalysed thiol exchange.

a, pH dependence. The activity of CcbV in 50 mM PIPES (pH 6.0-7.0) or 50 mM Tris-HCl (pH 7.5–9.0) buffer was measured. b, c, Determination of the steady-state kinetic parameters for substrate 4 and for MSH, respectively. The error bars are standard error of mean (n = 3).

Extended Data Figure 7 Characterization of LmbT-catalysed reverse and forward glycosylation.

For HPLC-MS analysis, the ESI m/z [M + H]+ modes are indicated in dashed rectangle. a, Examination of the acylated C8-sugar transfer in the presence of LmbT, which showed that LmbT was unable to utilize 4 as a substrate for reverse glycosylation to generate the predicted GDP-D-α-d-sugar 7 (left) in the absence (top right) and in the presence (lower right) of LmbT. b, Characterization of LmbT-catalysed forward glycosylation. LmbT used 3 as a substrate for glycosylation to generate the GDP-d-α-d-sugar 5 (left) in the absence (top right) and in the presence (lower right) of LmbT.

Extended Data Figure 8 Characterization of PPL incorporation in lincomycin A biosynthesis.

For HPLC-MS analysis, the ESI m/z [M + H]+ modes are indicated in the dashed rectangle. a, In vivo product profiles of S. lincolnensis strains, including the mutants (i, for ΔlmbC; ii, for ΔlmbN; iii, for ΔlmbD; and iv, for ΔlmbT) and the wild-type control (v). b, Process of the incorporation of PPL (with EGT S-conjugate 5) into intermediate 4. PCP (blue), peptidyl carrier protein; and I (grey), isomerase. c, HPLC-MS examination of LmbC-catalysed conversion of holo-LmbN-PCP (m/z [M + H]+ calculated 11,842.94, found 11,843.42) into PPL-acylated LmbN-PCP (m/z [M + H]+ calculated 11,982.01, found 11,983.21) in the absence (left) and in the presence (right) of ATP. d, In vitro analysis of the condensation between PPL and 5 to generate 4. The catalyst systems included LmbC + CcbD (i), LmbC + LmbN (ii), LmbC + LmbN-PCP (iii), LmbC + LmbN + CcbD (iv), and LmbC + LmbN-PCP + CcbD (v).

Extended Data Figure 9 Kinetic analysis of LmbT-catalysed reversible glycosylation.

a, pH dependence. The activity of LmbT in 50 mM PIPES (pH 6.0–7.0) or 50 mM Tris-HCl (pH 7.5–9.0) buffer was measured. b, c, Determination of the steady-state kinetic parameters for substrate 5 and for GDP, respectively, in LmbT-catalysed reverse glycosylation. The error bars are standard error of mean (n = 3). d, Determination of the equilibrium constant (Keq) of LmbT-catalysed glycosylation. Keq = ([GDP]/[3]) × ([5]/[EGT]) = 1.94 × 1 = 1.94.

Extended Data Figure 10 Validation of the highly ordered process involving EGT-mediated assembly and MSH-associated post-modifications in lincomycin biosynthesis.

For HPLC-MS analysis, the ESI m/z [M + H]+ modes are indicated in the dashed rectangle. a, Examination of the thiol exchange using 5 as a substrate in the absence (top right) and in the presence (bottom right) of CcbV, showing that CcbV was able to convert 5 to MSH S-conjugate 6 (left). b, Determination of the hydrolysis reaction in the absence (top right) and in the presence (bottom right) of LmbE, showing that this enzyme was unable to utilize 6 as a substrate and convert it to the predicted mercapturic acid 8 (left).

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Supplementary Data

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Supplementary Data

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Zhao, Q., Wang, M., Xu, D. et al. Metabolic coupling of two small-molecule thiols programs the biosynthesis of lincomycin A. Nature 518, 115–119 (2015).

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