Site-directed mutagenesis for cysteine residues of cobalt-containing nitrile hydratase

Modeling catalytic mechanism of nitrile hydratase by semi-empirical quantum mechanical calculation

“…Residue a-CEA 113 plays an important role for activating the active center, which is in consistent with experimental results of Hashimoto et al that site-directed mutagenesis of this residue would inactivate the enzyme [11]. Residue a-SER 112 contributes in two aspects, firstly, its side chain -OH can generate a hydrogen bond interacting with the N-bound hydrogen atom in the AM precursor, consequently helping the initial release of the product from the active center; secondly, it participates in the process of proton-rearrangement ensuring a fast and efficient transformation of the intermediate into the final product.…”

“…In the conserved active center with the common motif of CYS-(THR/SER)-LEU-CSD-SER-CEA, CYS was post-translationally modified into the oxidized forms of CSD, cysteine sulfuric acid (aCys111-SO 2 H) and CEA, cysteine sulfenic acid (aCys113-SOH), respectively [8][9][10]. Site-directed mutagenesis confirmed that these three cysteine residues are essential for active expression of the cobalt-containing H-NHase (high molecular mass-nitrile hydratase) [11].…”

Cobalt-requiring 5′-deoxy-5′-methylthioadenosine nucleosidase from soybean (Glycine max) cotyledon

“…DTNB modification and cobalt release were indicated by absorbance change at 412 nm (closed circle) and at 510 nm (open square), respectively cobalt/subunit, which is consistent with the result of above PAR analysis. It was previously reported that site-directed mutagenesis in cysteinyl residues of nitrile hydratase led to the release of cobalt ion and the loss of enzymatic activity (Hashimoto et al 2002). Cobalt promotes many growth processes in plant (Palit et al 1994).…”

Structure of Thiocyanate Hydrolase: A New Nitrile Hydratase Family Protein with a Novel Five-coordinate Cobalt(III) Center

“…We named the structure "claw setting". 6 Various studies using inhibitors, 9 recombinant enzymes, [10][11][12][13][14] and model compounds mimicking the metallocenters [15][16][17][18][19][20] have been performed, and it was demonstrated that these modifications are responsible for the catalytic activity. 9,10,19 However, their detailed functions remain unknown and the mechanism of biogenesis of the cysteine modifications has not been clarified.…”