Crystal Structure of StaL, a Glycopeptide Antibiotic Sulfotransferase from Streptomyces toyocaensis

Shi, Rong; Lamb, Sherry S.; Bhat, Sathesh; Sulea, Traian; Wright, Gerard D.; Matte, Allan; Cygler, Mirosław

doi:10.1074/jbc.m611912200

Cited by 23 publications

(20 citation statements)

References 60 publications

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“…The ternary complex crystallized in a different space group from all previously reported crystal forms of StaL (9). As in the earlier structures, StaL forms dimers (Fig.…”

Section: Resultsmentioning

confidence: 77%

“…We have previously speculated that the long loop α12/α13 (aa 217-235) that is disordered in the apo and PAP-bound structures would be involved in aglycone substrate binding and become ordered in its presence (9). However, this loop remains largely disordered in the present structure, with the exception of a short Leu226-Val229 segment located in the middle of this loop, which could be unambiguously traced in the electron density maps but only in subunit A. Reinvestigation of the final electron density maps for the apo or PAP-bound StaL structures (PDB ID codes 2OV8 and 2OVF) showed the presence of some positive density in this region but only in the PAP-bound structure, suggesting that ordering of this segment is associated already with PAP binding and strengthened by the DSA-binding event.…”

Section: Resultsmentioning

confidence: 99%

“…Structural elucidation of the DSA-binding mode in StaL helps explain the effect of mutations on enzymatic activity reported in our previous studies (9). The E205A mutant has only 4% of WT enzyme activity.…”

Section: Structure-based Rationalization Of Mutagenesis Data and Subsmentioning

confidence: 99%

“…We previously performed structure-function studies of two transferases involved in GPA modifications: the sulfotransferase StaL, from the biosynthetic pathway of the teicoplanin class antibiotic A47934 (9), and the methyltransferase MtfA, involved in the biosynthesis of the vancomycin class antibiotic chloroeremomycin (10). The sulfotransferase in particular offers a unique grafting of an anionic group with the capacity to modify biological activity.…”

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confidence: 99%

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Sulfonation of glycopeptide antibiotics by sulfotransferase StaL depends on conformational flexibility of aglycone scaffold

Shi

Munger

Kalan

et al. 2012

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

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Although glycopeptide antibiotics (GPAs), including vancomycin and teicoplanin, represent the most important class of antiinfective agents in the treatment of serious Gram-positive bacterial infections, their usefulness is threatened by the emergence of resistant strains. GPAs are complex natural products consisting of a heptapeptide skeleton assembled via nonribosomal peptide synthesis and constrained through multiple crosslinks, with diversity resulting from enzymatic modifications by a variety of tailoring enzymes, which can be used to produce GPA analogues that could overcome antibiotic resistance. GPA-modifying sulfotransferases are promising tools for generating the unique derivatives. Despite significant sequence and structural similarities, these sulfotransferases modify distinct side chains on the GPA scaffold. To provide insight into the spatial diversity of modifications, we have determined the crystal structure of the ternary complex of bacterial sulfotransferase StaL with the cofactor product 3′-phosphoadenosine 5′-phosphate and desulfo-A47934 aglycone substrate. Desulfo-A47934 binds with the hydroxyl group on the 4-hydroxyphenylglycine in residue 1 directed toward the 3′-phosphoadenosine 5′-phosphate and hydrogen-bonded to the catalytic His67. Homodimeric StaL can accommodate GPA substrate in only one of the two active sites because of potential steric clashes. Importantly, the aglycone substrate demonstrates a flattened conformation, in contrast to the cup-shaped structures observed previously. Analysis of the conformations of this scaffold showed that despite the apparent rigidity due to crosslinking between the side chains, the aglycone scaffold displays substantial flexibility, important for enzymatic modifications by the GPA-tailoring enzymes. We also discuss the potential of using the current structural information in generating unique GPA derivatives.substrate binding | substrate flexibility | substrate recognition

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“…The ternary complex crystallized in a different space group from all previously reported crystal forms of StaL (9). As in the earlier structures, StaL forms dimers (Fig.…”

Section: Resultsmentioning

confidence: 77%

Section: Resultsmentioning

confidence: 99%

Section: Structure-based Rationalization Of Mutagenesis Data and Subsmentioning

confidence: 99%

mentioning

confidence: 99%

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Sulfonation of glycopeptide antibiotics by sulfotransferase StaL depends on conformational flexibility of aglycone scaffold

Shi

Munger

Kalan

et al. 2012

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

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“…The teicoplanin aglycone is readily available and therefore we elected to use this closely related substrate in in vitro studies with the TEG sulfotransferases. The 3 sulfotransferases (Teg12, 13, and 14) in the TEG cluster are homologs of StaL, a 3Ј-phosphoadenosine 5Ј-phosphosulfate (PAPS)-dependent sulfotransferase found in the A47934 glycopeptide gene cluster (19). StaL has been used to generate monosulfated glycopeptides in vitro (20).…”

Section: Resultsmentioning

confidence: 99%

Cloning and characterization of new glycopeptide gene clusters found in an environmental DNA megalibrary

Banik

Brady

2008

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

105

100

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Glycopeptide antibiotics have long served as drugs of last resort for the treatment of antibiotic-resistant Gram-positive bacterial infections. Resistance to the clinically relevant glycopeptides, vancomycin and teicoplanin, threatens to undermine the usefulness of this important class of antibiotics. DNA extracted from a geographically diverse collection of soil samples was screened by PCR for the presence of sequences related to OxyC, an oxidative coupling enzyme found in glycopeptide biosynthetic gene clusters. Every soil sample examined contained at least 1 unique OxyC gene sequence. In an attempt to access the biosynthetic gene clusters associated with these OxyC sequences, a 10,000,000-membered environmental DNA (eDNA) megalibrary was created from a single soil sample. Two unique glycopeptide gene clusters were recovered from this eDNA megalibrary. Using the teicoplanin aglycone and the 3 sulfotransferases found in one of these gene clusters, mono-, di-, and trisulfated glycopeptide congeners were produced. The high frequency with which OxyC genes were found in environmental samples indicates that soil eDNA libraries are likely to be a rewarding source of glycopeptide gene clusters. Enzymes found in these gene clusters should be useful for generating new glycopeptides analogs. Environmental DNA megalibraries, like the one constructed for this study, can provide access to many of the natural product biosynthetic gene clusters that are predicted to be present in soil microbiomes.antibiotics ͉ eDNA ͉ metagenomics ͉ natural products ͉ uncultured bacteria

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Identification and Characterization of the Carbapenem MM 4550 and its Gene Cluster in Streptomyces argenteolus ATCC 11009

Lloyd

Moshos

et al. 2014

ChemBioChem

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Nearly 50 naturally-occurring carbapenem β-lactam antibiotics, most produced by Streptomyces, have been identified. The structural diversity of these compounds is limited to variance of the C-2 and C-6 side chains as well as the stereochemistry at C-5/C-6. These structural motifs are of interest both for their antibiotic effects and their biosynthesis. While the thienamycin gene cluster is the only active gene cluster publically available in this group, more comparative information is needed to understand the genetic basis of these structural differences. We report here the identification of MM 4550, a member of the olivanic acids, as the major carbapenem produced by S. argenteolus ATCC 11009. Its gene cluster was also identified by degenerate PCR and targeted gene inactivation. Sequence analysis revealed that genes encoding the biosynthesis of the bicyclic core and the C-6 and C-2 side chains are well conserved in the MM 4550 and thienamycin gene clusters. Three new genes, cmmSu, cmm17 and cmmPah were found in the new cluster and their putative functions in the sulfonation and epimerization of MM 4550 are proposed. Gene inactivation showed that, in addition to cmmI, two new genes, cmm22/23, encode a two-component response system thought to regulate the production of MM 4550. Overexpression of cmmI, cmm22 and cmm23 promoted MM 4550 production in an engineered strain. Finally, the involvement and putative roles of all genes in the MM 4550 cluster are proposed based on the results of bioinformatics analysis, gene inactivation, and analysis of disruption mutants. Overall, the differences between the thienamycin and MM 4550 gene clusters are reflected in characteristic structural elements and provide new insights into the biosynthesis of the complex carbapenems.

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Crystal Structure of StaL, a Glycopeptide Antibiotic Sulfotransferase from Streptomyces toyocaensis

Cited by 23 publications

References 60 publications

Sulfonation of glycopeptide antibiotics by sulfotransferase StaL depends on conformational flexibility of aglycone scaffold

Sulfonation of glycopeptide antibiotics by sulfotransferase StaL depends on conformational flexibility of aglycone scaffold

Cloning and characterization of new glycopeptide gene clusters found in an environmental DNA megalibrary

Identification and Characterization of the Carbapenem MM 4550 and its Gene Cluster in Streptomyces argenteolus ATCC 11009

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