Understanding the role of histidine in the GHSxG acyltransferase active site motif: evidence for histidine stabilization of the malonyl-enzyme intermediate. 2014

Sean Poust, and Isu Yoon, and Paul D Adams, and Leonard Katz, and Christopher J Petzold, and Jay D Keasling
Department of Chemical and Biomolecular Engineering, University of California, Berkeley, California, United States of America.

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.

UI MeSH Term Description Entries
D008958 Models, Molecular Models used experimentally or theoretically to study molecular shape, electronic properties, or interactions; includes analogous molecules, computer-generated graphics, and mechanical structures. Molecular Models,Model, Molecular,Molecular Model
D009154 Mutation Any detectable and heritable change in the genetic material that causes a change in the GENOTYPE and which is transmitted to daughter cells and to succeeding generations. Mutations
D004795 Enzyme Stability The extent to which an enzyme retains its structural conformation or its activity when subjected to storage, isolation, and purification or various other physical or chemical manipulations, including proteolytic enzymes and heat. Enzyme Stabilities,Stabilities, Enzyme,Stability, Enzyme
D006639 Histidine An essential amino acid that is required for the production of HISTAMINE. Histidine, L-isomer,L-Histidine,Histidine, L isomer,L-isomer Histidine
D000215 Acylation The addition of an organic acid radical into a molecule.
D000217 Acyltransferases Enzymes from the transferase class that catalyze the transfer of acyl groups from donor to acceptor, forming either esters or amides. (From Enzyme Nomenclature 1992) EC 2.3. Acyltransferase
D016297 Mutagenesis, Site-Directed Genetically engineered MUTAGENESIS at a specific site in the DNA molecule that introduces a base substitution, or an insertion or deletion. Mutagenesis, Oligonucleotide-Directed,Mutagenesis, Site-Specific,Oligonucleotide-Directed Mutagenesis,Site-Directed Mutagenesis,Site-Specific Mutagenesis,Mutageneses, Oligonucleotide-Directed,Mutageneses, Site-Directed,Mutageneses, Site-Specific,Mutagenesis, Oligonucleotide Directed,Mutagenesis, Site Directed,Mutagenesis, Site Specific,Oligonucleotide Directed Mutagenesis,Oligonucleotide-Directed Mutageneses,Site Directed Mutagenesis,Site Specific Mutagenesis,Site-Directed Mutageneses,Site-Specific Mutageneses
D020134 Catalytic Domain The region of an enzyme that interacts with its substrate to cause the enzymatic reaction. Active Site,Catalytic Core,Catalytic Region,Catalytic Site,Catalytic Subunit,Reactive Site,Active Sites,Catalytic Cores,Catalytic Domains,Catalytic Regions,Catalytic Sites,Catalytic Subunits,Core, Catalytic,Cores, Catalytic,Domain, Catalytic,Domains, Catalytic,Reactive Sites,Region, Catalytic,Regions, Catalytic,Site, Active,Site, Catalytic,Site, Reactive,Sites, Active,Sites, Catalytic,Sites, Reactive,Subunit, Catalytic,Subunits, Catalytic
D020816 Amino Acid Motifs Three-dimensional protein structural elements that are composed of a combination of secondary structures. They include HELIX-LOOP-HELIX MOTIFS and ZINC FINGERS. Motifs are typically the most conserved regions of PROTEIN DOMAINS and are critical for domain function. However, the same motif may occur in proteins or enzymes with different functions. AA Motifs,Motifs, Amino Acid,Protein Motifs,Protein Structure, Supersecondary,Supersecondary Protein Structure,AA Motif,Amino Acid Motif,Motif, AA,Motif, Amino Acid,Motif, Protein,Motifs, AA,Motifs, Protein,Protein Motif,Protein Structures, Supersecondary,Supersecondary Protein Structures

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