Cloning, sequencing, and heterologous expression of an Erwinia cypripedii 314B lactonase specific for l-α-hydroxyglutaric acid γ-lactone

• Published in: Applied microbiology and biotechnology 2006-08, Vol.71 (6), p.863-869
• Main Author: Mochizuki, Kazuya
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; amino acids; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biological and medical sciences; Biotechnology; Carboxylic Ester Hydrolases - chemistry; Carboxylic Ester Hydrolases - genetics; Carboxylic Ester Hydrolases - metabolism; Cloning, Molecular - methods; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; DNA, Bacterial - isolation & purification; enzyme activity; Erwinia; Erwinia - enzymology; Erwinia - genetics; Escherichia coli; Fundamental and applied biological sciences. Psychology; Gene Expression - genetics; gene overexpression; genes; Glutarates - chemistry; Glutarates - metabolism; Hydrolysis; Lactones - chemistry; Lactones - metabolism; Molecular Sequence Data; open reading frames; Pectobacterium cypripedii; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; sequence analysis; Sequence Analysis, DNA; signal peptide; Substrate Specificity
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-005-0224-2

The gene for a lactonase that stereospecifically hydrolyzes (S)-5-oxo-2-tetrahydrofurancarboxylic acid to l-α-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 ~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 ~35% similarity with a putative 3-carboxymuconate cyclase, but exhibited no such activity. The enzyme also showed ~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.