Cloning, sequencing, and heterologous expression of an Erwinia cypripedii 314B lactonase specific for L-alpha-hydroxyglutaric acid gamma-lactone.
The gene for a lactonase that stereospecifically hydrolyzes (S)-5-oxo-2-tetrahydrofurancarboxylic acid to L-alpha-hydroxyglutaric acid was isolated from Erwinia cypripedii 314B. Determination of the nucleotide sequence showed that the gene consists of a single open reading frame of 1,152 bp that encodes a 383-amino-acid protein. Comparison of the sequence of the predicted protein to that of the enzyme purified from E. cypripedii 314B revealed an N-terminal signal sequence of 19 amino acids. The gene for the mature enzyme was inserted into a pET vector and overexpressed in Escherichia coli. Active recombinant enzyme accumulated in the cells to approximately 30% of the total protein, and the enzyme was purified to homogeneity. The physical and catalytic properties of the recombinant enzyme were indistinguishable from those of the protein purified from E. cypripedii 314B. The deduced amino acid sequence displayed approximately 35% similarity with a putative 3-carboxymuconate cyclase, but exhibited no such activity. The enzyme also showed approximately 35% similarity with 6-phosphogluconolactonase. However, the activity of the enzyme toward 6-phosphogluconolactone was less than 2% of that toward (S)-5-oxo-2-tetrahydrofurancarboxylic acid, demonstrating a novel specificity for this lactonase.
Mochizuki
Kazuya
K
Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8566, Japan. mochizuki-kazuya@aist.go.jp
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004885:Erwinia / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression / Q000235:genetics*; D005977:Glutarates / Q000737:chemistry / Q000378:metabolism; D006868:Hydrolysis; D007783:Lactones / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
C108959:3-hydroxyglutaric acid; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D005977:Glutarates; D007783:Lactones; D011994:Recombinant Proteins; D002265:Carboxylic Ester Hydrolases; C020579:gluconolactonase