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Title: Substrate Recognition and Catalysis by the Cofactor-Independent Dioxygenase DpgC+

Abstract

The enzyme DpgC belongs to a small class of oxygenases not dependent on accessory cofactors for activity. DpgC is in the biosynthetic pathway for the nonproteinogenic amino acid 3, 5-dihydroxyphenylglycine in actinomycetes bacteria responsible for the production of the vancomycin/teicoplanin family of antibiotic natural products. The X-ray structure of DpgC confirmed the absence of cofactors and defined a novel hydrophobic dioxygen binding pocket adjacent to a bound substrate analogue. In this paper, the role specific amino acids play in substrate recognition and catalysis is examined through biochemical and structural characterization of site-specific enzyme mutations and alternate substrates. The results establish the importance of three amino acids, Arg254, Glu299, and Glu189, in the chemistry of DpgC. Arg254 and Glu189 join to form a specific contact with one of the phenolic hydroxyls of the substrate, and this interaction plays a key role in both substrate recognition and catalysis. The X-ray crystal structure of Arg254Lys was determined to address the role this residue plays in the chemistry. In addition, characterization of alternate substrate analogues demonstrates the presence and position of phenol groups are necessary for both enzyme recognition and downstream oxidation chemistry. Overall, this work defines the mechanism of substrate recognition and specificitymore » by the cofactor-independent dioxygenase DpgC.« less

Authors:
; ;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
959805
Report Number(s):
BNL-82791-2009-JA
Journal ID: ISSN 0006-2960; BICHAW; TRN: US201016%%949
DOE Contract Number:
DE-AC02-98CH10886
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemistry; Journal Volume: 46
Country of Publication:
United States
Language:
English
Subject:
36 MATERIALS SCIENCE; AMINO ACIDS; ANTIBIOTICS; BACTERIA; CATALYSIS; CHEMISTRY; CRYSTAL STRUCTURE; ENZYMES; MUTATIONS; OXIDATION; OXYGENASES; PHENOL; PRODUCTION; RESIDUES; SPECIFICITY; SUBSTRATES; national synchrotron light source

Citation Formats

Fielding,E., Widboom, P., and Bruner, S. Substrate Recognition and Catalysis by the Cofactor-Independent Dioxygenase DpgC+. United States: N. p., 2007. Web. doi:10.1021/bi701148b.
Fielding,E., Widboom, P., & Bruner, S. Substrate Recognition and Catalysis by the Cofactor-Independent Dioxygenase DpgC+. United States. doi:10.1021/bi701148b.
Fielding,E., Widboom, P., and Bruner, S. Mon . "Substrate Recognition and Catalysis by the Cofactor-Independent Dioxygenase DpgC+". United States. doi:10.1021/bi701148b.
@article{osti_959805,
title = {Substrate Recognition and Catalysis by the Cofactor-Independent Dioxygenase DpgC+},
author = {Fielding,E. and Widboom, P. and Bruner, S.},
abstractNote = {The enzyme DpgC belongs to a small class of oxygenases not dependent on accessory cofactors for activity. DpgC is in the biosynthetic pathway for the nonproteinogenic amino acid 3, 5-dihydroxyphenylglycine in actinomycetes bacteria responsible for the production of the vancomycin/teicoplanin family of antibiotic natural products. The X-ray structure of DpgC confirmed the absence of cofactors and defined a novel hydrophobic dioxygen binding pocket adjacent to a bound substrate analogue. In this paper, the role specific amino acids play in substrate recognition and catalysis is examined through biochemical and structural characterization of site-specific enzyme mutations and alternate substrates. The results establish the importance of three amino acids, Arg254, Glu299, and Glu189, in the chemistry of DpgC. Arg254 and Glu189 join to form a specific contact with one of the phenolic hydroxyls of the substrate, and this interaction plays a key role in both substrate recognition and catalysis. The X-ray crystal structure of Arg254Lys was determined to address the role this residue plays in the chemistry. In addition, characterization of alternate substrate analogues demonstrates the presence and position of phenol groups are necessary for both enzyme recognition and downstream oxidation chemistry. Overall, this work defines the mechanism of substrate recognition and specificity by the cofactor-independent dioxygenase DpgC.},
doi = {10.1021/bi701148b},
journal = {Biochemistry},
number = ,
volume = 46,
place = {United States},
year = {Mon Jan 01 00:00:00 EST 2007},
month = {Mon Jan 01 00:00:00 EST 2007}
}
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