The biosynthesis of nitrogen-, sulfur-, and high-carbon chain-containing sugars

Lin, Chia I.; McCarty, Reid M.; Liu, Hung‐wen

doi:10.1039/c2cs35438a

Cited by 81 publications

(90 citation statements)

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“…Despite recent advances made in unusual sugars biosynthesis research, little is known about thiosugar formation due to their rarity in natural products and the limited knowledge of sulphur incorporation in secondary metabolites 1,2,10,11 . In our studies of the biosynthesis of 2-thiosugar-containing antibiotic BE-7585A ( 1 , Fig.…”

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confidence: 99%

Co-opting sulphur-carrier proteins from primary metabolic pathways for 2-thiosugar biosynthesis

Sasaki

Zhang

Sun

et al. 2014

Nature

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Sulphur is an essential element for life and exists ubiquitously in living systems1,2. Yet, how the sulphur atom is incorporated in many sulphur-containing secondary metabolites remains poorly understood. For C-S bond formation in primary metabolites, the major ionic sulphur sources are the protein-persulphide and protein-thiocarboxylate3,4. In each case, the persulphide and thiocarboxylate group on these sulphur-carrier (donor) proteins are post-translationally generated through the action of a specific activating enzyme. In all bacterial cases reported thus far, the genes encoding the enzyme that catalyzes the actual C-S bond formation reaction and its cognate sulphur-carrier protein co-exist in the same gene cluster5. To study 2-thiosugar production in BE-7585A, an antibiotic from Amycolatopsis orientalis, we identified a putative 2-thioglucose synthase, BexX, whose protein sequence and mode of action appear similar to those of ThiG, the enzyme catalyzing thiazole formation in thiamin biosynthesis6,7. However, no sulphur-carrier protein gene could be located in the BE-7585A cluster. Subsequent genome sequencing revealed the presence of a few sulphur-carrier proteins likely involved in the biosynthesis of primary metabolites, but surprisingly only a single activating enzyme gene in the entire genome of A. orientalis. Further experiments showed that this activating enzyme is capable of adenylating each of these sulphur-carrier proteins, and likely also catalyzing the subsequent thiolation taking advantage of its rhodanese activity. A proper combination of these sulphur delivery systems is effective for BexX-catalyzed 2-thioglucose production. The ability of BexX to selectively distinguish sulphur-carrier proteins is given a structural basis using X-ray crystallography. These studies represent the first complete characterization of a thiosugar formation in nature and also demonstrate the receptor promiscuity of the sulphur-delivery system in A. orientalis. Our results also provide evidence that exploitation of sulphur-delivery machineries of primary metabolism for the biosynthesis of sulphur-containing natural products is likely a general strategy found in nature.

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Co-opting sulphur-carrier proteins from primary metabolic pathways for 2-thiosugar biosynthesis

Sasaki

Zhang

Sun

et al. 2014

Nature

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“…1 and 3) with similar k cat /K m values ( Table 1). The observed K m values of LnmJ-SH, CaJ-SH, and MaJ-SH with all tested substrates (1)(2)(3)(4)(5)(6)(7)(8)(9)(10) are between 1.9 ± 0.2 and 42 ± 6 mM (Table 1), which are too high to be relevant under the physiological reactions. These high K m values may result from the structural difference between the native substrates of the SH domains and the substrate mimics tested, the fact that the SH domains were studied in isolation (i.e., in truncation lacking the context of other domains within the native PKS modules), or a combination of both (Fig.…”

Section: Discussionmentioning

confidence: 97%

“…Recent literature has witnessed a flurry of reports on natural product biosynthesis featuring C-S bond formation or C-S bond cleavage steps (6)(7)(8)(9)(10)(11)(12)(13)(14)(15). Thus, as summarized in SI Appendix, Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Sulfur-containing metabolites are well known from both primary and secondary metabolism (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). The best-characterized sulfur-incorporating enzymes from primary metabolism include the following: (i) the group I and II cysteine desulfurases that use L-cysteine as the substrate and catalyze the production of a protein-bound persulfide and alanine, as exemplified by NifS, IscC, and CsdB (1-3); (ii) the D-cysteine desulfhydrases that use D-cysteine as the substrate and catalyze the β-elimination reaction to produce hydrogen sulfide, ammonia, and pyruvate (32); and (iii) the cysteine S-conjugate β-lyases that use L-cysteine-S-conjugates as the substrates and catalyze the β-elimination reaction to produce a thiol, ammonia, and pyruvate (33).…”

Section: Discussionmentioning

confidence: 99%

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C-S bond cleavage by a polyketide synthase domain

Lohman

Liu

et al. 2015

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

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Leinamycin (LNM) is a sulfur-containing antitumor antibiotic featuring an unusual 1,3-dioxo-1,2-dithiolane moiety that is spiro-fused to a thiazole-containing 18-membered lactam ring. The 1,3-dioxo-1,2-dithiolane moiety is essential for LNM's antitumor activity, by virtue of its ability to generate an episulfonium ion intermediate capable of alkylating DNA. We have previously cloned and sequenced the lnm gene cluster from Streptomyces atroolivaceus S-140. In vivo and in vitro characterizations of the LNM biosynthetic machinery have since established that: (i) the 18-membered macrolactam backbone is synthesized by LnmP, LnmQ, LnmJ, LnmI, and LnmG, (ii) the alkyl branch at C-3 of LNM is installed by LnmK, LnmL, LnmM, and LnmF, and (iii) leinamycin E1 (LNM E1), bearing a thiol moiety at C-3, is the nascent product of the LNM hybrid nonribosomal peptide synthetase (NRPS)-acyltransferase (AT)-less type I polyketide synthase (PKS). Sulfur incorporation at C-3 of LNM E1, however, has not been addressed. Here we report that: (i) the bioinformatics analysis reveals a pyridoxal phosphate (PLP)-dependent domain, we termed cysteine lyase (SH) domain (LnmJ-SH), within PKS module-8 of LnmJ; (ii) the LnmJ-SH domain catalyzes C-S bond cleavage by using L-cysteine and L-cysteine S-modified analogs as substrates through a PLP-dependent β-elimination reaction, establishing L-cysteine as the origin of sulfur at C-3 of LNM; and (iii) the LnmJ-SH domain, sharing no sequence homology with any other enzymes catalyzing C-S bond cleavage, represents a new family of PKS domains that expands the chemistry and enzymology of PKSs and might be exploited to incorporate sulfur into polyketide natural products by PKS engineering.cysteine metabolism | enzyme mechanism | genome mining | pathway engineering | sulfur metabolism S ulfur is found in many primary metabolites, such as thiamin, biotin, molybdenum cofactors, lipoic acid, iron-sulfur clusters, and nucleosides including 2-thiocytidine, 4-thiouridine, and 2-methylthio-N 6 -isopentenyl adenosine (1). There is a wealth of information on how sulfur is incorporated into these primary metabolites, the key step of which is catalyzed by a desulfurase to produce a protein-bound cysteine persulfide by using cysteine as a substrate (2-5). Sulfur is also found in many secondary metabolites (interchangeably referred to as natural products here). The major groups of sulfur-containing natural products are peptides produced by ribosome or nonribosomal peptide synthetases (NRPSs). Sulfur incorporation into these natural products from intact cysteine or methionine is well characterized. However, sulfur incorporation into other natural products remains poorly understood. Only a few cases featured by C-S bond formation or C-S bond cleavage steps were reported to date, and most of the mechanisms require further investigation (6-15).Leinamycin (LNM) is a sulfur-containing antitumor antibiotic produced by Streptomyces atroolivaceus S-140 (16). The structure of LNM is characterized by an unusual 1,3-dioxo-1,2-dith...

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“…We demonstrate that the hypothetical protein is a novel lytic transglycosylase and shares parallel chemistry to enzymes of Gram-negative cell wall recycling pathways (Lee et al, 2013). Structurally diverse oligosaccharides can target a wide range of biological systems, underlying their potential pharmacological value (McCranie and Bachmann, 2014), rare sugars are important metabolic building blocks of natural products (Lin et al, 2013), and 1,6-anhydro sugars are commonly utilized for the laboratory synthesis of glycosylated molecules (Tanaka et al, 2009). This example illustrates that atypical pathways represent an avenue for the discovery of new biocatalytic chemistry.…”

Section: Introductionmentioning

confidence: 99%

An Atypical Orphan Carbohydrate-NRPS Genomic Island Encodes a Novel Lytic Transglycosylase

Guo

Crawford

2014

Chemistry & Biology

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SUMMARY Microbial genome sequencing platforms have produced a deluge of orphan biosynthetic pathways suspected of biosynthesizing new small molecules with pharmacological relevance. Genome synteny analysis provides an assessment of genomic island content, which is enriched in natural product gene clusters. Here we identified an atypical orphan carbohydrate-nonribosomal peptide synthetase (NRPS) genomic island in Photorhabdus luminescens using genome synteny analysis. Heterologous expression of the pathway led to the characterization of five new oligosaccharide metabolites with lysozyme inhibitory activities. The oligosaccharides harbor a 1,6-anhydro-β-D-N-acetyl-glucosamine (Glc-NAc) moiety, a rare structural feature for natural products. Gene deletion analysis and biochemical reconstruction of oligosaccharide production led to the discovery that a hypothetical protein in the pathway is a novel lytic transglycosylase responsible for bicyclic sugar formation. The example presented here supports a notion where targeting select genomic islands with a reduced reliance on known protein homologies could enhance the discovery of new metabolic chemistry and biology.

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The biosynthesis of nitrogen-, sulfur-, and high-carbon chain-containing sugars

Cited by 81 publications

References 209 publications

Co-opting sulphur-carrier proteins from primary metabolic pathways for 2-thiosugar biosynthesis

Co-opting sulphur-carrier proteins from primary metabolic pathways for 2-thiosugar biosynthesis

C-S bond cleavage by a polyketide synthase domain

An Atypical Orphan Carbohydrate-NRPS Genomic Island Encodes a Novel Lytic Transglycosylase

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