Understanding the role of histidine in the GHSxG acyltransferase active site motif: Evidence for histidine stabilization of the malonyl-enzyme intermediate
Abstract
Acyltransferases determine which extender units are incorporated into polyketide and fatty acid products. Thus, the ping-pong acyltransferase mechanism utilizes a serine in a conserved GHSxG motif. However, the role of the conserved histidine in this motif is poorly understood. We observed that a histidine to alanine mutation (H640A) in the GHSxG motif of the malonyl-CoA specific yersiniabactin acyltransferase results in an approximately seven-fold higher hydrolysis rate over the wildtype enzyme, while retaining transacylation activity. We propose two possibilities for the reduction in hydrolysis rate: either H640 structurally stabilizes the protein by hydrogen bonding with a conserved asparagine in the ferredoxin-like subdomain of the protein, or a water-mediated hydrogen bond between H640 and the malonyl moiety stabilizes the malonyl-O-AT ester intermediate.
- Authors:
-
- Univ. of California, Berkeley, CA (United States)
- Joint BioEnergy Institute, Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
- Synthetic Biology Engineering Research Center, Emeryville, CA (United States); Joint BioEnergy Institute, Emeryville, CA (United States)
- Univ. of California, Berkeley, CA (United States); Synthetic Biology Engineering Research Center, Emeryville, CA (United States); Joint BioEnergy Institute, Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
- Publication Date:
- Research Org.:
- Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
- Sponsoring Org.:
- USDOE Advanced Research Projects Agency - Energy (ARPA-E); USDOE Office of Science (SC), Biological and Environmental Research (BER)
- OSTI Identifier:
- 1211510
- Grant/Contract Number:
- 0000206-1577; AC02-05CH11231
- Resource Type:
- Accepted Manuscript
- Journal Name:
- PLoS ONE
- Additional Journal Information:
- Journal Volume: 9; Journal Issue: 10; Journal ID: ISSN 1932-6203
- Publisher:
- Public Library of Science
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; hydrolysis; histidine; sequence motif analysis; hydrogen bonding; serine; alanine; esters; fatty acids
Citation Formats
Poust, Sean, Yoon, Isu, Adams, Paul D., Katz, Leonard, Petzold, Christopher J., and Keasling, Jay D. Understanding the role of histidine in the GHSxG acyltransferase active site motif: Evidence for histidine stabilization of the malonyl-enzyme intermediate. United States: N. p., 2014.
Web. doi:10.1371/journal.pone.0109421.
Poust, Sean, Yoon, Isu, Adams, Paul D., Katz, Leonard, Petzold, Christopher J., & Keasling, Jay D. Understanding the role of histidine in the GHSxG acyltransferase active site motif: Evidence for histidine stabilization of the malonyl-enzyme intermediate. United States. https://doi.org/10.1371/journal.pone.0109421
Poust, Sean, Yoon, Isu, Adams, Paul D., Katz, Leonard, Petzold, Christopher J., and Keasling, Jay D. Mon .
"Understanding the role of histidine in the GHSxG acyltransferase active site motif: Evidence for histidine stabilization of the malonyl-enzyme intermediate". United States. https://doi.org/10.1371/journal.pone.0109421. https://www.osti.gov/servlets/purl/1211510.
@article{osti_1211510,
title = {Understanding the role of histidine in the GHSxG acyltransferase active site motif: Evidence for histidine stabilization of the malonyl-enzyme intermediate},
author = {Poust, Sean and Yoon, Isu and Adams, Paul D. and Katz, Leonard and Petzold, Christopher J. and Keasling, Jay D.},
abstractNote = {Acyltransferases determine which extender units are incorporated into polyketide and fatty acid products. Thus, the ping-pong acyltransferase mechanism utilizes a serine in a conserved GHSxG motif. However, the role of the conserved histidine in this motif is poorly understood. We observed that a histidine to alanine mutation (H640A) in the GHSxG motif of the malonyl-CoA specific yersiniabactin acyltransferase results in an approximately seven-fold higher hydrolysis rate over the wildtype enzyme, while retaining transacylation activity. We propose two possibilities for the reduction in hydrolysis rate: either H640 structurally stabilizes the protein by hydrogen bonding with a conserved asparagine in the ferredoxin-like subdomain of the protein, or a water-mediated hydrogen bond between H640 and the malonyl moiety stabilizes the malonyl-O-AT ester intermediate.},
doi = {10.1371/journal.pone.0109421},
journal = {PLoS ONE},
number = 10,
volume = 9,
place = {United States},
year = {Mon Oct 06 00:00:00 EDT 2014},
month = {Mon Oct 06 00:00:00 EDT 2014}
}
Free Publicly Available Full Text
Publisher's Version of Record
Other availability
Search WorldCat to find libraries that may hold this journal
Cited by: 7 works
Citation information provided by
Web of Science
Web of Science
Save to My Library
You must Sign In or Create an Account in order to save documents to your library.
Works referenced in this record:
Catalysis, Specificity, and ACP Docking Site of Streptomyces coelicolor Malonyl-CoA:ACP Transacylase
journal, February 2003
- Keatinge-Clay, Adrian T.; Shelat, Anang A.; Savage, David F.
- Structure, Vol. 11, Issue 2
The Escherichia coli Malonyl-CoA:Acyl Carrier Protein Transacylase at 1.5-Å Resolution. : CRYSTAL STRUCTURE OF A FATTY ACID SYNTHASE COMPONENT
journal, June 1995
- Serre, Laurence; Verbree, Elizabeth C.; Dauter, Zbigniew
- Journal of Biological Chemistry, Vol. 270, Issue 22
The Pfam protein families database
journal, January 2004
- Bateman, Alex; Coin, Lachlan; Durbin, Richard
- Nucleic Acids Research, Vol. 32, Issue S1, p. D138-D141
Malonyl-CoA:ACP Transacylase from Streptomyces coelicolor Has Two Alternative Catalytically Active Nucleophiles
journal, September 2001
- Dreier, Juerg; Li, Qing; Khosla, Chaitan
- Biochemistry, Vol. 40, Issue 41
Kinetic and Mechanistic Analysis of the Malonyl CoA:ACP Transacylase from Streptomyces coelicolor Indicates a Single Catalytically Competent Serine Nucleophile at the Active Site †
journal, February 2002
- Szafranska, Anna E.; Hitchman, Timothy S.; Cox, Russell J.
- Biochemistry, Vol. 41, Issue 5
Yersiniabactin Synthetase
journal, March 2002
- Miller, Deborah Ann; Luo, Lusong; Hillson, Nathan
- Chemistry & Biology, Vol. 9, Issue 3
A continuous coupled enzyme assay for bacterial malonyl–CoA:acyl carrier protein transacylase (FabD)
journal, August 2003
- Molnos, Juliette; Gardiner, Rana; Dale, Glenn E.
- Analytical Biochemistry, Vol. 319, Issue 1
Mechanism and Specificity of an Acyltransferase Domain from a Modular Polyketide Synthase
journal, March 2013
- Dunn, Briana J.; Cane, David E.; Khosla, Chaitan
- Biochemistry, Vol. 52, Issue 11
Absolute metabolite concentrations and implied enzyme active site occupancy in Escherichia coli
journal, June 2009
- Bennett, Bryson D.; Kimball, Elizabeth H.; Gao, Melissa
- Nature Chemical Biology, Vol. 5, Issue 8
Accessing natural product biosynthetic processes by mass spectrometry
journal, October 2008
- Bumpus, Stefanie B.; Kelleher, Neil L.
- Current Opinion in Chemical Biology, Vol. 12, Issue 5
Crystal Structure of the Acyltransferase Domain of the Iterative Polyketide Synthase in Enediyne Biosynthesis
journal, May 2012
- Liew, Chong Wai; Nilsson, Martina; Chen, Ming Wei
- Journal of Biological Chemistry, Vol. 287, Issue 27
Skylign: a tool for creating informative, interactive logos representing sequence alignments and profile hidden Markov models
journal, January 2014
- Wheeler, Travis J.; Clements, Jody; Finn, Robert D.
- BMC Bioinformatics, Vol. 15, Issue 1
Kinetic and Mechanistic Analysis of the Malonyl CoA:ACP Transacylase from Streptomyces coelicolor Indicates a Single Catalytically Competent Serine Nucleophile at the Active Site †
journal, February 2002
- Szafranska, Anna E.; Hitchman, Timothy S.; Cox, Russell J.
- Biochemistry, Vol. 41, Issue 5
Mechanism and Specificity of an Acyltransferase Domain from a Modular Polyketide Synthase
journal, March 2013
- Dunn, Briana J.; Cane, David E.; Khosla, Chaitan
- Biochemistry, Vol. 52, Issue 11
Absolute metabolite concentrations and implied enzyme active site occupancy in Escherichia coli
journal, June 2009
- Bennett, Bryson D.; Kimball, Elizabeth H.; Gao, Melissa
- Nature Chemical Biology, Vol. 5, Issue 8
The Pfam protein families database
journal, January 2004
- Bateman, Alex; Coin, Lachlan; Durbin, Richard
- Nucleic Acids Research, Vol. 32, Issue S1, p. D138-D141
Works referencing / citing this record:
Control Mechanism for cis Double‐Bond Formation by Polyunsaturated Fatty‐Acid Synthases
journal, February 2019
- Hayashi, Shohei; Satoh, Yasuharu; Ogasawara, Yasushi
- Angewandte Chemie International Edition, Vol. 58, Issue 8
Structural and Biochemical Insight into the Recruitment of Acyl Carrier Protein‐Linked Extender Units in Ansamitocin Biosynthesis
journal, January 2020
- Zhang, Fa; Ji, Huining; Ali, Imtiaz
- ChemBioChem, Vol. 21, Issue 9
Control Mechanism for cis Double-Bond Formation by Polyunsaturated Fatty-Acid Synthases
journal, January 2019
- Hayashi, Shohei; Satoh, Yasuharu; Ogasawara, Yasushi
- Angewandte Chemie, Vol. 131, Issue 8