title: 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. 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.affiliation: Department of Chemical and Biomolecular Engineering, University of California, Berkeley, California, United States of America.
authors.forename: Sean
authors.initials: S
authors.lastname: Poust
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of California, Berkeley, California, United States of America.
authors.forename: Isu
authors.initials: I
authors.lastname: Yoon
authors.affiliation: Joint BioEnergy Institute, Emeryville, California, United States of America; Physical Bioscience division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America.
authors.forename: Paul D
authors.initials: PD
authors.lastname: Adams
authors.affiliation: Synthetic Biology Engineering Research Center, Emeryville, California, United States of America; Joint BioEnergy Institute, Emeryville, California, United States of America.
authors.forename: Leonard
authors.initials: L
authors.lastname: Katz
authors.affiliation: Joint BioEnergy Institute, Emeryville, California, United States of America; Physical Bioscience division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Petzold
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of California, Berkeley, California, United States of America; QB3 Institute, University of California, Berkeley, California, United States of America; Synthetic Biology Engineering Research Center, Emeryville, California, United States of America; Joint BioEnergy Institute, Emeryville, California, United States of America; Physical Bioscience division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America.
authors.forename: Jay D
authors.initials: JD
authors.lastname: Keasling
mesh_terms: D000215:Acylation; D000217:Acyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs; D020134:Catalytic Domain; D004795:Enzyme Stability; D006639:Histidine; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation
chemical_list: D006639:Histidine; D000217:Acyltransferases