Role of conserved histidine residues in D-aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6

Mamoru Wakayama; Harutaka Yada; Shun Ichi Kanda; Shin Ichi Hayashi; Yukinori Yatsuda; Kenji Sakai; Mitsuaki Moriguchi

doi:10.1271/bbb.64.1

Role of conserved histidine residues in D-aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6

Mamoru Wakayama, Harutaka Yada, Shun Ichi Kanda, Shin Ichi Hayashi, Yukinori Yatsuda, Kenji Sakai, Mitsuaki Moriguchi

Research output: Contribution to journal › Article › peer-review

16 Citations (Scopus)

Abstract

D-Aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) was strongly inactivated by diethylpyrocarbonate (DEPC). An H67N mutant was barely active, with a k_cat/K_m 6.3 × 10⁴ times lower than that of the recombinant wild-type enzyme, while the H67I mutant lost detectable activity. The H67N mutant had almost constant K_m, but greatly decreased k_cat. These results suggested that His67 is essential to the catalytic event. Both H69N and H69I mutants were overproduced in the insoluble fraction. The k_cat/K_m of H250N mutant was reduced by a factor of 2.5 × 10⁴-old as compared with the wild-type enzyme. No significant difference between H251N mutant and wild-type enzymes in the K_m and k_cat was found. The Zn content of H250N mutant was nearly half of that of wild-type enzyme. These results suggest that the His250 residue might be essential to catalysis via Zn binding.

Original language	English
Pages (from-to)	1-8
Number of pages	8
Journal	Bioscience, Biotechnology and Biochemistry
Volume	64
Issue number	1
DOIs	https://doi.org/10.1271/bbb.64.1
Publication status	Published - Jan 2000
Externally published	Yes

All Science Journal Classification (ASJC) codes

Biotechnology
Analytical Chemistry
Biochemistry
Applied Microbiology and Biotechnology
Molecular Biology
Organic Chemistry

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title = "Role of conserved histidine residues in D-aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6",

abstract = "D-Aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) was strongly inactivated by diethylpyrocarbonate (DEPC). An H67N mutant was barely active, with a kcat/Km 6.3 × 104 times lower than that of the recombinant wild-type enzyme, while the H67I mutant lost detectable activity. The H67N mutant had almost constant Km, but greatly decreased kcat. These results suggested that His67 is essential to the catalytic event. Both H69N and H69I mutants were overproduced in the insoluble fraction. The kcat/Km of H250N mutant was reduced by a factor of 2.5 × 104-old as compared with the wild-type enzyme. No significant difference between H251N mutant and wild-type enzymes in the Km and kcat was found. The Zn content of H250N mutant was nearly half of that of wild-type enzyme. These results suggest that the His250 residue might be essential to catalysis via Zn binding.",

author = "Mamoru Wakayama and Harutaka Yada and Kanda, {Shun Ichi} and Hayashi, {Shin Ichi} and Yukinori Yatsuda and Kenji Sakai and Mitsuaki Moriguchi",

year = "2000",

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doi = "10.1271/bbb.64.1",

language = "English",

volume = "64",

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journal = "Bioscience, Biotechnology and Biochemistry",

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AU - Wakayama, Mamoru

AU - Yada, Harutaka

AU - Kanda, Shun Ichi

AU - Hayashi, Shin Ichi

AU - Yatsuda, Yukinori

AU - Sakai, Kenji

AU - Moriguchi, Mitsuaki

PY - 2000/1

Y1 - 2000/1

N2 - D-Aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) was strongly inactivated by diethylpyrocarbonate (DEPC). An H67N mutant was barely active, with a kcat/Km 6.3 × 104 times lower than that of the recombinant wild-type enzyme, while the H67I mutant lost detectable activity. The H67N mutant had almost constant Km, but greatly decreased kcat. These results suggested that His67 is essential to the catalytic event. Both H69N and H69I mutants were overproduced in the insoluble fraction. The kcat/Km of H250N mutant was reduced by a factor of 2.5 × 104-old as compared with the wild-type enzyme. No significant difference between H251N mutant and wild-type enzymes in the Km and kcat was found. The Zn content of H250N mutant was nearly half of that of wild-type enzyme. These results suggest that the His250 residue might be essential to catalysis via Zn binding.

AB - D-Aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) was strongly inactivated by diethylpyrocarbonate (DEPC). An H67N mutant was barely active, with a kcat/Km 6.3 × 104 times lower than that of the recombinant wild-type enzyme, while the H67I mutant lost detectable activity. The H67N mutant had almost constant Km, but greatly decreased kcat. These results suggested that His67 is essential to the catalytic event. Both H69N and H69I mutants were overproduced in the insoluble fraction. The kcat/Km of H250N mutant was reduced by a factor of 2.5 × 104-old as compared with the wild-type enzyme. No significant difference between H251N mutant and wild-type enzymes in the Km and kcat was found. The Zn content of H250N mutant was nearly half of that of wild-type enzyme. These results suggest that the His250 residue might be essential to catalysis via Zn binding.

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Role of conserved histidine residues in D-aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6

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