title: Structure-based modification of D-alanine-D-alanine ligase from Thermotoga maritima ATCC 43589 for depsipeptide synthesis.
abstract: Depsipeptides are peptide-like polymers consisting of amino acids and hydroxy acids, and are expected to be new functional materials for drug-delivery systems and polymer science. In our previous study, D-alanyl-D-lactate, a type of depsipeptide, was enzymatically synthesized using D-alanine-D-alanine ligase from Thermotoga maritima ATCC 43589 (TmDdl) by Y207F substitution. Thereafter, in this study, further mutagenesis was introduced, based on structural comparison between TmDdl and a well-characterized D-alanine-D-alanine ligase from Escherichia coli. The S137A/Y207F mutant showed higher D-alanyl-D-lactate and lower D-alanyl-D-alanine synthesizing activity than the Y207F mutant. This suggests that substitution at the S137 residue contributes to product selectivity. Saturated mutagenesis on S137 revealed that the S137G/Y207F mutant showed the highest D-alanyl-D-lactate synthesizing activity. Moreover, the mutant showed broad substrate specificity toward D-amino acid and recognized D-lactate and D,L-isoserine as substrates. On the basis of these characteristics, various depsipeptides can be produced using S137G/Y207F-replaced TmDdl.
authors.affiliation: Department of Applied Chemistry, School of Advanced Science and Engineering, Faculty of Science and Engineering, Waseda University, Japan.
authors.forename: Tomoki
authors.initials: T
authors.lastname: Nakagawa
authors.forename: Ryoko
authors.initials: R
authors.lastname: Satake
authors.forename: Masaru
authors.initials: M
authors.lastname: Sato
authors.forename: Kuniki
authors.initials: K
authors.lastname: Kino
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D047630:Depsipeptides / Q000096:biosynthesis*; D004926:Escherichia coli / Q000201:enzymology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010453:Peptide Synthases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D020124:Thermotoga maritima / Q000201:enzymology*
chemical_list: D047630:Depsipeptides; D010453:Peptide Synthases; C019638:D-alanylalanine synthetase