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Title: Substrate and Product Complexes of Escherichia Coli Adenylosuccinate Lyase Provide New Insights into the Enzymatic Mechanism

Abstract

Adenylosuccinate lyase (ADL) catalyzes the breakdown of 5-aminoimidazole- (N-succinylocarboxamide) ribotide (SAICAR) to 5-aminoimidazole-4-carboxamide ribotide (AICAR) and fumarate, and of adenylosuccinate (ADS) to adenosine monophosphate (AMP) and fumarate in the de novo purine biosynthetic pathway. ADL belongs to the argininosuccinate lyase (ASL)/fumarase C superfamily of enzymes. Members of this family share several common features including: a mainly {alpha}-helical, homotetrameric structure; three regions of highly conserved amino acid residues; and a general acid-base catalytic mechanism with the overall {beta}-elimination of fumarate as a product. The crystal structures of wild-type Escherichia coli ADL (ec-ADL), and mutant-substrate (H171A-ADS) and -product (H171N-AMP{center_dot}FUM) complexes have been determined to 2.0, 1.85, and 2.0 {angstrom} resolution, respectively. The H171A-ADS and H171N-AMP{center_dot}FUM structures provide the first detailed picture of the ADL active site, and have enabled the precise identification of substrate binding and putative catalytic residues. Contrary to previous suggestions, the ec-ADL structures implicate S295 and H171 in base and acid catalysis, respectively. Furthermore, structural alignments of ec-ADL with other superfamily members suggest for the first time a large conformational movement of the flexible C3 loop (residues 287-303) in ec-ADL upon substrate binding and catalysis, resulting in its closure over the active site. This loop movement has been observedmore » in other superfamily enzymes, and has been proposed to be essential for catalysis. The ADL catalytic mechanism is re-examined in light of the results presented here.« less

Authors:
; ; ; ; ;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
930465
Report Number(s):
BNL-81217-2008-JA
Journal ID: ISSN 0022-2836; JMOBAK; TRN: US200904%%733
DOE Contract Number:  
DE-AC02-98CH10886
Resource Type:
Journal Article
Resource Relation:
Journal Name: Journal of Molecular Biology; Journal Volume: 370; Journal Issue: 3
Country of Publication:
United States
Language:
English
Subject:
36 MATERIALS SCIENCE; AMINO ACIDS; AMP; BREAKDOWN; CATALYSIS; CLOSURES; CRYSTAL STRUCTURE; ENZYMES; ESCHERICHIA COLI; LYASES; PURINES; RESIDUES; RESOLUTION; SUBSTRATES; national synchrotron light source

Citation Formats

Tsai,M., Koo, J., Yip, P., Colman, R., Segall, M., and Howell, P. Substrate and Product Complexes of Escherichia Coli Adenylosuccinate Lyase Provide New Insights into the Enzymatic Mechanism. United States: N. p., 2007. Web. doi:10.1016/j.jmb.2007.04.052.
Tsai,M., Koo, J., Yip, P., Colman, R., Segall, M., & Howell, P. Substrate and Product Complexes of Escherichia Coli Adenylosuccinate Lyase Provide New Insights into the Enzymatic Mechanism. United States. doi:10.1016/j.jmb.2007.04.052.
Tsai,M., Koo, J., Yip, P., Colman, R., Segall, M., and Howell, P. Mon . "Substrate and Product Complexes of Escherichia Coli Adenylosuccinate Lyase Provide New Insights into the Enzymatic Mechanism". United States. doi:10.1016/j.jmb.2007.04.052.
@article{osti_930465,
title = {Substrate and Product Complexes of Escherichia Coli Adenylosuccinate Lyase Provide New Insights into the Enzymatic Mechanism},
author = {Tsai,M. and Koo, J. and Yip, P. and Colman, R. and Segall, M. and Howell, P.},
abstractNote = {Adenylosuccinate lyase (ADL) catalyzes the breakdown of 5-aminoimidazole- (N-succinylocarboxamide) ribotide (SAICAR) to 5-aminoimidazole-4-carboxamide ribotide (AICAR) and fumarate, and of adenylosuccinate (ADS) to adenosine monophosphate (AMP) and fumarate in the de novo purine biosynthetic pathway. ADL belongs to the argininosuccinate lyase (ASL)/fumarase C superfamily of enzymes. Members of this family share several common features including: a mainly {alpha}-helical, homotetrameric structure; three regions of highly conserved amino acid residues; and a general acid-base catalytic mechanism with the overall {beta}-elimination of fumarate as a product. The crystal structures of wild-type Escherichia coli ADL (ec-ADL), and mutant-substrate (H171A-ADS) and -product (H171N-AMP{center_dot}FUM) complexes have been determined to 2.0, 1.85, and 2.0 {angstrom} resolution, respectively. The H171A-ADS and H171N-AMP{center_dot}FUM structures provide the first detailed picture of the ADL active site, and have enabled the precise identification of substrate binding and putative catalytic residues. Contrary to previous suggestions, the ec-ADL structures implicate S295 and H171 in base and acid catalysis, respectively. Furthermore, structural alignments of ec-ADL with other superfamily members suggest for the first time a large conformational movement of the flexible C3 loop (residues 287-303) in ec-ADL upon substrate binding and catalysis, resulting in its closure over the active site. This loop movement has been observed in other superfamily enzymes, and has been proposed to be essential for catalysis. The ADL catalytic mechanism is re-examined in light of the results presented here.},
doi = {10.1016/j.jmb.2007.04.052},
journal = {Journal of Molecular Biology},
number = 3,
volume = 370,
place = {United States},
year = {Mon Jan 01 00:00:00 EST 2007},
month = {Mon Jan 01 00:00:00 EST 2007}
}