ISBN: 978-83-60043-10-3 - eurobic9

Eurobic9, 2-6 September, 2008, Wrocław, Poland
P142. Maturation Mechanism of a New Nitrile Hydratase Family Protein,
Thiocyanate Hydrolase
M. Odaka a , S. Hori a , T. Arakawa a , H. Nakayama b , N. Dohmae b , H. Mino c ,
Y. Katayama d , M. Yohda a
a
Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16
Naka-cho, 184-8588, Koganei, Japan,
b
Biomolecular Characterization Team, RIKEN, 2-1 Hirosawa, 351-0198, Wako, Japan
c
Division.of Material Science (Physics), Nagoya University, Chikusa, 464-8602, Nagoya, Japan
d
Department of Environmental and Natural Resource S, Tokyo University of Agriculture and Technology, 3-5-8
Saiwaicho, 183-8509, Fuchu, Japan
e-mail: modaka@cc.tuat.ac.jp
Nitrile hydratase (NHase) family proteins are Co- or Fe-containing enzymes having two post-translationally
modified Cys ligands, Cys-SO2H and Cys-SOH. NHase family proteins require their specific activator proteins
for the functional expression. Here, we studied the function of P15K, the activator protein of a new Co-type
NHase family protein, thiocyanate hydrolase (SCNase). SCNase catalyzes the hydrolysis of thiocyanate to
carbonyl sulfide and ammonia. It consists of α, β and γ subunits and has a hetero-dodecamer structure, (αβγ)4.
When P15K was co-expressed with each SCNase subunit in E. coli, only γsubunit which had the Co-binding site
formed a stable 1: 1 complex (γP15K). In contrast, the isolated γ subunit (γ(+Co)) as well as γP15K
(γ(+Co)P15K) was obtained when they were co-expressed in the Co-enriched medium. γ(+Co) as well as
γ(+Co)P15K incorporated stoichiometric amounts of Co ions and possessed the Cys-SO2H modification like
native SCNase, suggesting that these complexes are intermediate species in the maturation systems of SCNase.
Then, we expressed SCNase (A) α, (B) β or (C) γ subunits in the presence of γ(+Co) using a cell free protein
synthesis system. In (A) and (C), the αβ or αβγ complexes were obtained, respectively, while no βγ complex was
detected in (B). Interestingly, only the reaction mixture of (C) exhibited the SCNase activity. Thus, we
concluded that γ(+Co) assembles with the α subunit subsequently with the β subunit, to form mature SCNase.
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