Subunit dissociation and stability alteration of d-hydantoinase deleted at the terminal amino acid residue

Abstract

Two variants of D-hydantoinase (HYD), created by deletion of one amino acid residue of at either the N-or C-terminus, were expressed in Escherichia coli and purified by twostep chromatography. Compared with HYD, HYDc1 with the C-terminal Arg deletion retained 43% activity, while HYDn1 with the N-terminal Ser deletion had no activity using DLHydantoin as substrate. Based on HYD dimer with a molecular weight of 103 kDa, HYDc1 is a monomer of 52 kDa and HYDn1 is a mixture of dimer and monomer. Moreover, HYDc1 displayed higher pH stability and lower thermal stability compared to HYD. In addition, the secondary and tertiary structures of HYDc1 were not significantly changed in contrast to the ones of HYDn1. All data imply that the C-terminal Arg of the HYD is crucial for homodimeric architecture of the enzyme, but non-essential for catalysis, while the N-terminal Ser is required for both conformation and catalysis of the enzyme.

Crystal Structures of Vertebrate Dihydropyrimidinase and Complexes from Tetraodon nigroviridis with Lysine Carbamylation

“…Previous reports have shown that modifications to the C termini of DHP/HYD enzymes could decrease the enzyme activity (9, 48,[53][54][55]. In the D-HYD from B. stearothermophilus and Bacillus thermocatenulatus, the truncated enzymes with a deletion of 40 amino acids on the C terminus have lost 64% of their enzymatic activities (48).…”

“…In the D-HYD from B. stearothermophilus and Bacillus thermocatenulatus, the truncated enzymes with a deletion of 40 amino acids on the C terminus have lost 64% of their enzymatic activities (48). In Pseudomonas putida YZ-26, a series of deletions were performed in the D-HYD and all of the truncated enzymes exhibited either low or non-detectable activities (53,54). However, CD spectroscopy showed that these deletions of the C-terminal fragment did not alter the secondary structures of the overall folding (48,54).…”

“…In Pseudomonas putida YZ-26, a series of deletions were performed in the D-HYD and all of the truncated enzymes exhibited either low or non-detectable activities (53,54). However, CD spectroscopy showed that these deletions of the C-terminal fragment did not alter the secondary structures of the overall folding (48,54). More recently, deletions on the C terminus of DHP from human, a vertebrate similar to T. nigroviridis, have been undertaken, and these mutants also show no detectable activity (9).…”

The Flexibility of the Non-Conservative Region at the C Terminus of d-Hydantoinase from Pseudomonas putida YZ-26 is Extremely Limited

“…Based on different enantioselectivities for various substrates, hydantoinases can be classified into three types: D-, L-or DL-configuration [3,4]. However, the D-hydantoinase from microorganisms has attracted much more attentions because it is used for the production of optically pure D-amino acids that are key intermediates in the synthesis of commercial products such as β-lactam semisynthetic antibiotics, peptides, hormones, pyretroids, and pesticides [5].…”

Quantitative analysis and functional evaluation of zinc ion in the d-hydantoinase from Pseudomonas putida YZ-26

“…Enzymatic assay D-Hydantoinase activity was measured by a colorimetric method (Niu et al 2007). An aliquot of the enzyme was added into the reaction mixture in a total volume of 1.5 ml, contained 100 mM hydantoin and 50 mM TB8.…”