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Title: Crystal Structure of StaL, A Glycopeptide Antibiotic Sulfotransferase from Streptomyces Toyocaensis

Abstract

Over the past decade, antimicrobial resistance has emerged as a major public health crisis. Glycopeptide antibiotics such as vanco-mycin and teicoplanin are clinically important for the treatment of Gram-positive bacterial infections. StaL is a 3'-phosphoadenosine 5'-phosphosulfate-dependent sulfotransferase capable of sulfating the cross-linked heptapeptide substrate both in vivo and in vitro, yielding the product A47934 [GenBank], unique teicoplanin-class glycopeptide antibiotic. The sulfonation reaction catalyzed by StaL constitutes the final step in A47934 [GenBank] biosynthesis. Here we report the crystal structure of StaL and its complex with the cofactor product 3'-phosphoadenosine 5'-phosphate. This is only the second prokaryotic sulfotransferase to be structurally characterized. StaL belongs to the large sulfotransferase family and shows higher similarity to cytosolic sulfotransferases (ST) than to the bacterial ST (Stf0). StaL has a novel dimerization motif, different from any other STs that have been structurally characterized. We have also applied molecular modeling to investigate the binding mode of the unique substrate, desulfo-A47934. Based on the structural analysis and modeling results, a series of residues was mutated and kinetically characterized. In addition to the conserved residues (Lys{sup 12}, His{sup 67}, and Ser{sup 98}), molecular modeling, fluorescence quenching experiments, and mutagenesis studies identified several other residues essential for substrate bindingmore » and/or activity, including Trp{sup 34}, His{sup 43}, Phe{sup 77}, Trp{sup 132}, and Glu{sup 205}.« less

Authors:
; ; ; ; ; ;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
929910
Report Number(s):
BNL-80495-2008-JA
Journal ID: ISSN 0021-9258; JBCHA3; TRN: US200822%%1085
DOE Contract Number:
DE-AC02-98CH10886
Resource Type:
Journal Article
Resource Relation:
Journal Name: Journal of Biological Chemistry; Journal Volume: 282
Country of Publication:
United States
Language:
English
Subject:
36 MATERIALS SCIENCE; ANTIBIOTICS; BACTERIAL DISEASES; BIOSYNTHESIS; CRYSTAL STRUCTURE; DIMERIZATION; FLUORESCENCE; IN VITRO; IN VIVO; MICROBIAL DRUG RESISTANCE; MUTAGENESIS; PUBLIC HEALTH; QUENCHING; RESIDUES; STREPTOMYCES; SUBSTRATES; SULFONATION; national synchrotron light source

Citation Formats

Shi,R., Lamb, S., Bhat, S., Sulea, T., Wright, G., Matte, A., and Cygler, M. Crystal Structure of StaL, A Glycopeptide Antibiotic Sulfotransferase from Streptomyces Toyocaensis. United States: N. p., 2007. Web. doi:10.1074/jbc.M611912200.
Shi,R., Lamb, S., Bhat, S., Sulea, T., Wright, G., Matte, A., & Cygler, M. Crystal Structure of StaL, A Glycopeptide Antibiotic Sulfotransferase from Streptomyces Toyocaensis. United States. doi:10.1074/jbc.M611912200.
Shi,R., Lamb, S., Bhat, S., Sulea, T., Wright, G., Matte, A., and Cygler, M. Mon . "Crystal Structure of StaL, A Glycopeptide Antibiotic Sulfotransferase from Streptomyces Toyocaensis". United States. doi:10.1074/jbc.M611912200.
@article{osti_929910,
title = {Crystal Structure of StaL, A Glycopeptide Antibiotic Sulfotransferase from Streptomyces Toyocaensis},
author = {Shi,R. and Lamb, S. and Bhat, S. and Sulea, T. and Wright, G. and Matte, A. and Cygler, M.},
abstractNote = {Over the past decade, antimicrobial resistance has emerged as a major public health crisis. Glycopeptide antibiotics such as vanco-mycin and teicoplanin are clinically important for the treatment of Gram-positive bacterial infections. StaL is a 3'-phosphoadenosine 5'-phosphosulfate-dependent sulfotransferase capable of sulfating the cross-linked heptapeptide substrate both in vivo and in vitro, yielding the product A47934 [GenBank], unique teicoplanin-class glycopeptide antibiotic. The sulfonation reaction catalyzed by StaL constitutes the final step in A47934 [GenBank] biosynthesis. Here we report the crystal structure of StaL and its complex with the cofactor product 3'-phosphoadenosine 5'-phosphate. This is only the second prokaryotic sulfotransferase to be structurally characterized. StaL belongs to the large sulfotransferase family and shows higher similarity to cytosolic sulfotransferases (ST) than to the bacterial ST (Stf0). StaL has a novel dimerization motif, different from any other STs that have been structurally characterized. We have also applied molecular modeling to investigate the binding mode of the unique substrate, desulfo-A47934. Based on the structural analysis and modeling results, a series of residues was mutated and kinetically characterized. In addition to the conserved residues (Lys{sup 12}, His{sup 67}, and Ser{sup 98}), molecular modeling, fluorescence quenching experiments, and mutagenesis studies identified several other residues essential for substrate binding and/or activity, including Trp{sup 34}, His{sup 43}, Phe{sup 77}, Trp{sup 132}, and Glu{sup 205}.},
doi = {10.1074/jbc.M611912200},
journal = {Journal of Biological Chemistry},
number = ,
volume = 282,
place = {United States},
year = {Mon Jan 01 00:00:00 EST 2007},
month = {Mon Jan 01 00:00:00 EST 2007}
}
  • The TEG gene cluster, a glycopeptide biosynthetic gene cluster that is predicted to encode the biosynthesis of a polysulfated glycopeptide congener, was recently cloned from DNA extracted directly from desert soil. This predicted glycopeptide gene cluster contains three closely related sulfotransferases (Teg12, -13, and -14) that sulfate teicoplanin-like glycopeptides at three unique sites. Here we report a series of structures: an apo structure of Teg12, Teg12 bound to the desulfated cosubstrate 3{prime}-phosphoadenosine 5{prime}-phosphate, and Teg12 bound to the teicoplanin aglycone. Teg12 appears to undergo a series of significant conformational rearrangements during glycopeptide recruitment, binding, and catalysis. Loop regions that exhibitmore » the most conformational flexibility show the least sequence conservation between TEG sulfotransferases. Site-directed mutagenesis guided by our structural studies confirmed the importance of key catalytic residues as well as the importance of residues found throughout the conformationally flexible loop regions.« less
  • The 2.7 Å resolution crystal structure of Teg14, a glycopeptide sulfotransferase cloned from an uncultured soil bacterium, is described. The relationship of Teg14 to other sulfotransferases is discussed. The TEG gene cluster was recently isolated from an environmental DNA library and is predicted to encode the biosynthesis of a polysulfated glycopeptide congener. Three closely related sulfotransferases found in the TEG gene cluster (Teg12, Teg13 and Teg14) have been shown to sulfate the teicoplanin aglycone at three unique sites. Crystal structures of the first sulfotransferase from the TEG cluster, Teg12, in complex with the teicoplanin aglycone and its desulfated cosubstrate PAPmore » have recently been reported [Bick et al. (2010 ▶), Biochemistry, 49, 4159–4168]. Here, the 2.7 Å resolution crystal structure of the apo form of Teg14 is reported. Teg14 sulfates the hydroxyphenylglycine at position 4 in the teicoplanin aglycone. The Teg14 structure is discussed and is compared with those of other bacterial 3′-phosphoadenosine 5′-phosphosulfate-dependent sulfotransferases facilitating crystallographic experiments, especially in the field of microcrystallography.« less
  • C-1027 is a chromoprotein enediyne antitumor antibiotic produced by Streptomyces globisporus. In the last step of biosynthesis of the (S)-3-chloro-5-hydroxy-beta-tyrosine moiety of the C-1027 enediyne chromophore, SgcE6 and SgcC compose a two-component monooxygenase that hydroxylates the C-5 position of (S)-3-chloro-beta-tyrosine. This two-component monooxygenase is remarkable for two reasons. (i) SgcE6 specifically reacts with FAD and NADH, and (ii) SgcC is active with only the peptidyl carrier protein (PCP)-tethered substrate. To address the molecular details of substrate specificity, we determined the crystal structures of SgcE6 and SgcC at 1.66 and 2.63 A resolution, respectively. SgcE6 shares a similar β-barrel fold withmore » the class I HpaC-like flavin reductases. A flexible loop near the active site of SgcE6 plays a role in FAD binding, likely by providing sufficient space to accommodate the AMP moiety of FAD, when compared to that of FMN-utilizing homologues. SgcC shows structural similarity to a few, other known FADH 2-dependent monooxygenases and sheds light on some biochemically but not structurally characterized homologues. In conclusion, the crystal structures reported here provide insights into substrate specificity, and comparison with homologues provides a catalytic mechanism of the two-component, FADH 2-dependent monooxygenase (SgcE6 and SgcC) that catalyzes the hydroxylation of a PCP-tethered substrate.« less
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