Catalytic analysis of a recombinant D-hydantoinase from Agrobacterium tumefaciens

• Published in: Biotechnology letters Vol. 25; no. 13; pp. 1067 - 1073
• Main Authors: CLEMENTE-JIMENEZ, Josefa Maria, MARTINEZ-ROGRIGUEZ, Sergio, MINGORANCE-CAZORLA, Lydia, DE LA ESCALERA-HUESO, Santiago, LAS HERAS-VAZQUEZ, Francisco Javier, RODRIGUEZ-VICO, Felipe
• Format: Journal Article
• Language: English
• Published: Dordrecht Springer 01.07.2003; Springer Nature B.V
• Subjects: Agrobacterium tumefaciens - chemistry; Agrobacterium tumefaciens - classification; Agrobacterium tumefaciens - enzymology; Agrobacterium tumefaciens - genetics; Amidohydrolases - biosynthesis; Amidohydrolases - chemistry; Amidohydrolases - genetics; Amidohydrolases - isolation & purification; Amino Acid Sequence; Amino acids; Biological and medical sciences; Catalysis; Cloning, Molecular; Coenzymes - chemistry; Enzymatic activity; Enzyme Activation; Enzyme Stability; Fundamental and applied biological sciences. Psychology; Hydrogen-Ion Concentration; Metals - chemistry; Molecular Sequence Data; Proteins; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Species Specificity; Substrate Specificity; Temperature; D-hydantoinase; Agrobacterium; expression; amino acids
• ISSN: 0141-5492; 1573-6776
• DOI: 10.1023/A:1024115220304

The D-hydantoinase gene of a wild strain of Agrobacterium tumefaciens BQL9 had 99.78% nucleotide sequence identity with other available Agrobacterium genes. The resulting amino acid sequence showed two important substitutions affecting two alpha-helixes in the secondary structure of the protein. The union of Mn2+ to the protein was essential for activating the enzyme and was independent of the temperature. D-Hydantoinase only was inactivated in the presence of 70 mM EDTA and at over 40 degrees C. The enzyme showed both hydantoinase and pyrimidinase activities, but only with the D-enantiomers of the substrates. Activity was greater for substrates with apolar groups in the number 5 carbon atom of the hydantoin. The native structure of the N-terminal end of this D-hydantoinase proved to be indispensable to its enzymatic activity.