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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

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:
 [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)
Publication Date:
OSTI Identifier:
1211510
Grant/Contract Number:
0000206-1577; AC02-05CH11231
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
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) (SC-23)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES hydrolysis; histidine; sequence motif analysis; hydrogen bonding; serine; alanine; esters; fatty acids