title: Isolation and amino acid sequence of a dehydratase acting on d-erythro-3-hydroxyaspartate from Pseudomonas sp. N99, and its application in the production of optically active 3-hydroxyaspartate.
abstract: An enzyme catalyzing the ammonia-lyase reaction for the conversion of d-erythro-3-hydroxyaspartate to oxaloacetate was purified from the cell-free extract of a soil-isolated bacterium Pseudomonas sp. N99. The enzyme exhibited ammonia-lyase activity toward l-threo-3-hydroxyaspartate and d-erythro-3-hydroxyaspartate, but not toward other 3-hydroxyaspartate isomers. The deduced amino acid sequence of the enzyme, which belongs to the serine/threonine dehydratase family, shows similarity to the sequence of l-threo-3-hydroxyaspartate ammonia-lyase (EC 4.3.1.16) from Pseudomonas sp. T62 (74%) and Saccharomyces cerevisiae (64%) and serine racemase from Schizosaccharomyces pombe (65%). These results suggest that the enzyme is similar to l-threo-3-hydroxyaspartate ammonia-lyase from Pseudomonas sp. T62, which does not act on d-erythro-3-hydroxyaspartate. We also then used the recombinant enzyme expressed in Escherichia coli to produce optically pure l-erythro-3-hydroxyaspartate and d-threo-3-hydroxyaspartate from the corresponding dl-racemic mixtures. The enzymatic resolution reported here is one of the simplest and the first enzymatic method that can be used for obtaining optically pure l-erythro-3-hydroxyaspartate.
authors.affiliation: a Laboratory of Microbial Physiology, Research Faculty of Agriculture , Hokkaido University , Sapporo , Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Nagano
authors.affiliation: a Laboratory of Microbial Physiology, Research Faculty of Agriculture , Hokkaido University , Sapporo , Japan.
authors.forename: Kana
authors.initials: K
authors.lastname: Shibano
authors.affiliation: a Laboratory of Microbial Physiology, Research Faculty of Agriculture , Hokkaido University , Sapporo , Japan.
authors.forename: Yu
authors.initials: Y
authors.lastname: Matsumoto
authors.affiliation: a Laboratory of Microbial Physiology, Research Faculty of Agriculture , Hokkaido University , Sapporo , Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Yokota
authors.affiliation: a Laboratory of Microbial Physiology, Research Faculty of Agriculture , Hokkaido University , Sapporo , Japan.
authors.forename: Masaru
authors.initials: M
authors.lastname: Wada
mesh_terms: D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006836:Hydro-Lyases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D009904:Optical Rotation; D062907:Oxaloacetic Acid / Q000378:metabolism*; D011485:Protein Binding; D011549:Pseudomonas / Q000737:chemistry / Q000201:enzymology*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000201:enzymology; D012568:Schizosaccharomyces / Q000737:chemistry / Q000201:enzymology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; C011274:3-hydroxyaspartic acid; D062907:Oxaloacetic Acid; D001224:Aspartic Acid; D006836:Hydro-Lyases
keywords: 3-hydroxyaspartate; Pseudomonas sp. N99; ammonia-lyase; enzymatic resolution; pyridoxal 5′-phosphate