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

Eurobic9, 2-6 September, 2008, Wrocław, Poland
P75. Structural Evidence for the Catalytic Mechanism of Nitrile Hydratase
Proposed by Time-resolved X-ray Crystallography Using a Novel
Substrate, tert-butylisonitrile
K. Hashimoto a , H. Suzuki b , K. Taniguchi c , M. Yohda a , T. Noguchi b , M. Odaka a
a
Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16
Naka-cho, 184 8588, Koganei, Tokyo, Japan
e-mail: kounda@bel.bio.tuat.ac.jp
b
Institute of Materials Science, University of Tsukuba, 1-1-1 Tennohdai, 305 8573, Tsukuba, Ibaraki, Japan
c
Eco-Soft Material Research Units, RIKEN, Wako, Saitama, 2-1 Hirosawa, 351 0198, Wako, Saitama, Japan
Nitrile hydratase (NHase) catalyzes the hydration of nitriles to amides. NHase of Rhodococcus sp. N771 has a
non-heme Fe catalytic center with two oxidized Cys ligands, αCys112-SO2H and αCys114-SOH. The metal is
proposed to function as a Lewis acid, but the detailed mechanism remains unclear. Recently, we found that
NHase catalyzes the conversion of tert-butylisonitrile (tBuNC) to tert-butylamine (tBuNH2). Here we present the
first structural evidence for the catalytic mechanism of NHase. When the reaction was monitored by ATR-FTIR
only tBuNH2 was observed, suggesting that the product from the isonitrile carbon escaped as a gas. The product
was identified as CO by being trapped with reduced hemoglobin. Thus, NHase catalyze the reaction of both
nitriles and isonitriles with one equivalent molar amount of water molecules. Crystals of the nitrosylated inactive
NHase were soaked with tBuNC. The catalytic reaction was initiated by photo-induced denitrosylation and
stopped by flash-cooling with nitrogen gas at elapsed times. tBuNC was first trapped at the hydrophobic pocket
above the Fe center and then coordinated to the Fe ion at 120 min. From 340 to 440 mins, the shapes of the
electron densities of tBuNC were significantly changed and a new electron density observed near the isonitrile
carbon as well as the sulfenate oxygen of αCys114. These results demonstrate that the substrate was coordinated
to the iron and then attacked by a water molecule activated by αCys114-SOH.
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