Molecular Biotechnology

, Volume 60, Issue 9, pp 690–697 | Cite as

Effect of Active Site Pocket Structure Modification of d-Stereospecific Amidohydrolase on the Recognition of Stereospecific and Hydrophobic Substrates

  • Yasmeen Yousif Ahmed Elyas
  • Kazusa Miyatani
  • Katsuhiko Shimizu
  • Jiro ArimaEmail author
Original Paper


d-Stereospecific amidohydrolase (DAH) from Streptomyces sp. 82F2 has potential utility for the synthesis of d/l configuration dipeptides by an aminolysis reaction. Structural comparison of DAH with substrate-bound d-amino acid amidase revealed that three residues located in the active site pocket of DAH (Thr145, Ala267, and Gly271) might be involved in interactions with d-phenylalanine substrate. We substituted Ala267 and Gly271, which are located at the bottom of the hydrophobic pocket of DAH, with Phe and observed changes in the stereoselectivity and specific activity toward the free and acetylated forms of d/l-Phe-methyl esters. In contrast, the mutation of Thr145, which likely supplies negative charge for recognition of the amino group of the substrate, hardly affected the stereoselectivity of the enzyme. A similar effect was observed in an investigation of hydrolysis and aminolysis reactions using the acetylated forms of d/l-Phe-methyl esters and 1,8-diaminooctane as an acyl-donor and acyl-acceptor, respectively. Substrate binding by DAH was disrupted by the mutation of Ala267 to Val or Trp and kinetic analysis showed that the hydrophobicity of the bottom of the active site pocket (Ala267 and Gly271) is important for both stereoselectivity and recognition of hydrophobic substrates.


d-Stereospecific amidohydrolase Stereospecificity Substrate recognition Hydrophobic pocket 



This research was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (No. 26450124). We thank Dr. T. Bito Faculty of Agriculture, Tottori University, for helpful advice and technical support during this study.

Supplementary material

12033_2018_104_MOESM1_ESM.doc (1.2 mb)
Supplementary material 1 (DOC 1212 KB)


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Yasmeen Yousif Ahmed Elyas
    • 1
  • Kazusa Miyatani
    • 2
  • Katsuhiko Shimizu
    • 3
  • Jiro Arima
    • 4
    Email author
  1. 1.United Graduate School of Agricultural SciencesTottori UniversityTottoriJapan
  2. 2.Graduate School of Agricultural SciencesTottori UniversityTottoriJapan
  3. 3.Division of Regional Contribution and Lifelong Learning, Organization of Regional Industrial-Academic CooperationTottori UniversityTottoriJapan
  4. 4.Department of Agricultural, Life and Environmental Sciences, Faculty of AgricultureTottori UniversityTottoriJapan

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