Elusive source of sulfur unravelled

The metabolic origin of the sulfur atom in the naturally occurring antibiotic lincomycin A has been obscure — until now. The biosynthetic steps involved reveal surprising roles for two sulfur-containing metabolites. See Letter p.115

Many vital biological molecules contain sulfur, the metabolic origins of which can be predicted fairly easily by those fluent in nature's biosynthetic language. But the sources of sulfur atoms are still difficult to predict for a few such molecules, including several with potentially useful biological activities, such as the anticancer agents calicheamicin1 and leinamycin2, and the antibacterial agent albomycin3. On page 115 of this issue, Zhao et al.4 report the three key enzymatic steps that together install the sulfur atom of the antibiotic lincomycin A (Fig. 1) during its biosynthesis in the bacterium Streptomyces lincolnensis. Using a combination of techniques, they reveal a new role for mycothiol (MSH) — a bacterial sulfur-containing antioxidant — as a sulfur donor. They also report the unprecedented involvement of ergothioneine (EGT), another sulfur-containing bacterial metabolite, in an enzyme-catalysed process: the chemical activation of the carbon atom that will eventually bear the sulfur.

Figure 1: Biosynthesis of lincomycin A.

Zhao et al.4 report the biosynthetic mechanism by which a sulfur atom is incorporated into the naturally occurring antibiotic lincomycin A. The two main components of the molecule are an octose sugar (orange) and a PPL unit (blue). a, In the biosynthetic pathway, the LmbT enzyme attaches ergothioneine (EGT, a bacterial metabolite) to the octose sugar by forming a new carbon–sulfur bond; a nucleotide (GDP) attached to the sugar is lost in the process. b, The enzymes LmbC, N and D attach PPL to the sugar. c, LmbV then replaces EGT with mycothiol (MSH, a bacterial antioxidant), forming a new carbon–sulfur bond; the sulfur atom from MSH is the one that ends up in the antibiotic. d, Finally, a multi-step process beginning with a reaction catalysed by LmbE converts the MSH group into the methylmercapto group (−SCH3) of lincomycin A.

Deciphering the details of a complex biosynthetic pathway is, in some ways, like solving a jigsaw puzzle that has many more pieces than are needed to construct the correct picture. The molecular pieces (enzymes and their encoding genes, substrates and products) must often be carefully examined to solve the puzzle successfully (determine the correct biosynthetic pathway). Some interesting mechanisms for incorporating sulfur into molecules have been revealed in the past few years, including those involved in the biosynthesis of thiamine5 and the antibiotic BE-7585A (ref. 6).

The addition of whole-genome sequencing and sophisticated comparative genomics to the biosynthetic chemist's extensive repertoire of techniques has helped researchers to solve complex biosynthetic puzzles. Specifically, the method helps them to predict the relationships between enzyme amino-acid sequences and functions more accurately, and allows easier identification of genes involved in supplying biosynthetic precursors. This is particularly evident in genes found at chromosomal positions distant from those of related biosynthetic genes, which, in bacteria, are often tightly clustered. Zhao et al. use the full complement of tools to discover the elusive origin of the sulfur atom in lincomycin A.

As is often done in biosynthetic studies, the authors began by working backwards through the proposed pathway, attempting to reconstruct its molecular logic by isolating intermediates that accumulate when the activities of specific genes are disrupted. They proposed that lmbE — a gene present in the sequenced biosynthetic gene cluster for lincomycin A, and which encodes a homologue of an amidase enzyme involved in an MSH-dependent detoxification process in certain bacteria — has a role in sulfur-atom incorporation.

Sure enough, when Zhao and colleagues disrupted the function of lmbE, they observed the accumulation of a lincomycin-like intermediate in which an MSH moiety was attached through its sulfur atom to the predicted sulfur-incorporation site. They also found that purified LmbE enzyme cleaves the amide bond of this intermediate, and that feeding either the isolated intermediate or the product of its cleavage by LmbE to an S. lincolnensis mutant in which the function of an MSH-regenerating gene, mshA, was disrupted led to restoration of lincomycin A production. The authors had identified mshA through genome sequencing and analysis as part of the study. These findings confirmed the function of LmbE and the intermediacy of its substrate and product in the biosynthetic pathway.

Next, the researchers examined the function of lmbV, another gene present in the lincomycin cluster. This gene is homologous to an enzyme that catalyses an MSH-dependent isomerization reaction. When Zhao and co-workers disrupted the function of lmbV, they observed the unexpected accumulation of another lincomycin-like intermediate, in which an EGT moiety is attached through its sulfur atom to the site occupied by MSH in the metabolite isolated from the lmbE mutant. The authors also identified the EGT-containing intermediate in their mshA-disruption mutant, which suggests that LmbV catalyses the replacement of the EGT moiety by MSH. They could not prove this directly, because they were not able to express and purify LmbV, but they confirmed their theory by showing that a homologue of LmbV — CcbV, an enzyme from a similar biosynthetic pathway7 — catalyses the proposed reaction. They further confirmed the key role of EGT in the biosynthesis of lincomycin A by disrupting the function of egtD, a gene involved in the biosynthesis of EGT that they again found through genome mining.

Finally, Zhao et al. tested the function of another enzyme in the lincomycin pathway, LmbT, which they thought might attach EGT to a biosynthetic intermediate. LmbT is a homologue of glycosyltransferase enzymes, which attach sugars to other molecules. The researchers performed a series of gene-disruption and in vitro biochemical experiments, establishing that LmbT must act before installation of the 4-propyl-L-proline (PPL) moiety, which forms part of the structure of lincomycin A. In the process, they also proved that three more enzymes — LmbC (ref. 8), LmbN and LmbD — collectively incorporate PPL into the antibiotic.

Zhao and colleagues went on to isolate the suspected product of LmbT and to demonstrate the enzyme's function using in vitro assays. They discovered that LmbT catalyses the transfer of lincomycin A's sugar (for which the biosynthetic pathway has previously been reported9) to EGT, thus chemically activating the sugar in readiness for its reaction with MSH later in the pathway. Such a role is completely unprecedented: EGT was known to exist as a metabolite, but not as a substrate for an enzyme-catalysed reaction.

A particularly impressive aspect of this work is the authors' use of an intricate series of in vivo and in vitro experiments that relied on intermediates obtained from mutant cultures and from both enzymatic and chemical syntheses, guided by comparative gene analysis and genome mining. More generally, the study demonstrates that integration of primary metabolites (those that are essential for an organism's survival, such as MSH and EGT) and secondary metabolites (non-essential compounds, such as the products of the Lmb enzymes) is crucial for the biosynthesis of complex molecules. It also highlights the ingenious ways in which nature repurposes enzymes — in this case, using homologues of MSH-dependent detoxification enzymes for biosynthesis. And the establishment of functions for LmbE, LmbV and LmbT will no doubt help researchers to work out the functions of the enzymes' numerous homologues in the ever-growing roster of sequenced genomes.

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Correspondence to Charles E. Melançon III.

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Melançon, C. Elusive source of sulfur unravelled. Nature 518, 45–46 (2015).

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