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Title: Understanding the role of histidine in the GHSxG acyltransferase active site motif: Evidence for histidine stabilization of the malonyl-enzyme intermediate

Journal Article · · PLoS ONE
 [1];  [1];  [2];  [3];  [2];  [4]
  1. Univ. of California, Berkeley, CA (United States)
  2. Joint BioEnergy Institute, Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
  3. Synthetic Biology Engineering Research Center, Emeryville, CA (United States); Joint BioEnergy Institute, Emeryville, CA (United States)
  4. 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)
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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.

Research Organization:
Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
Sponsoring Organization:
USDOE Advanced Research Projects Agency - Energy (ARPA-E); USDOE Office of Science (SC), Biological and Environmental Research (BER)
Grant/Contract Number:
0000206-1577; AC02-05CH11231
OSTI ID:
1211510
Journal Information:
PLoS ONE, Vol. 9, Issue 10; ISSN 1932-6203
Publisher:
Public Library of ScienceCopyright Statement
Country of Publication:
United States
Language:
English
Citation Metrics:
Cited by: 7 works
Citation information provided by
Web of Science

References (12)

Catalysis, Specificity, and ACP Docking Site of Streptomyces coelicolor Malonyl-CoA:ACP Transacylase journal February 2003
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
The Pfam protein families database journal January 2004
Malonyl-CoA:ACP Transacylase from Streptomyces coelicolor Has Two Alternative Catalytically Active Nucleophiles journal September 2001
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
Yersiniabactin Synthetase journal March 2002
A continuous coupled enzyme assay for bacterial malonyl–CoA:acyl carrier protein transacylase (FabD) journal August 2003
Mechanism and Specificity of an Acyltransferase Domain from a Modular Polyketide Synthase journal March 2013
Absolute metabolite concentrations and implied enzyme active site occupancy in Escherichia coli journal June 2009
Accessing natural product biosynthetic processes by mass spectrometry journal October 2008
Crystal Structure of the Acyltransferase Domain of the Iterative Polyketide Synthase in Enediyne Biosynthesis journal May 2012
Skylign: a tool for creating informative, interactive logos representing sequence alignments and profile hidden Markov models journal January 2014

Cited By (3)

Control Mechanism for cis Double‐Bond Formation by Polyunsaturated Fatty‐Acid Synthases journal February 2019
Structural and Biochemical Insight into the Recruitment of Acyl Carrier Protein‐Linked Extender Units in Ansamitocin Biosynthesis journal January 2020
Control Mechanism for cis Double-Bond Formation by Polyunsaturated Fatty-Acid Synthases journal January 2019

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Related Subjects

59 BASIC BIOLOGICAL SCIENCES
hydrolysis
histidine
sequence motif analysis
hydrogen bonding
serine
alanine
esters
fatty acids