Identification of crucial histidines involved in carbon-nitrogen triple bond synthesis by aldoxime dehydratase.
Aldoxime dehydratase (OxdA), which is a novel heme protein, catalyzes the dehydration of an aldoxime to a nitrile even in the presence of water in the reaction mixture. The combination of site-directed mutagenesis of OxdA (mutation of all conserved histidines in the aldoxime dehydratase superfamily), estimation of the heme contents and specific activities of the mutants, and CD and resonance Raman spectroscopic analyses led to the identification of the proximal and distal histidines in this unique enzyme. The heme contents and CD spectra in the far-UV region of all mutants except for the H299A one were almost identical to those of the wild-type OxdA, whereas the H299A mutant lost the ability of binding heme, demonstrating that His(299) is the proximal histidine. On the other hand, substitution of alanine for His(320) did not affect the overall structure of OxdA but caused loss of its ability of carbon-nitrogen triple bond synthesis and a lower shift of the Fe-C stretching band in the resonance Raman spectrum for the CO-bound form. Furthermore, the pH dependence of the wild-type OxdA closely followed the His protonation curves observed for other proteins. These findings suggest that His(320) is located in the distal heme pocket of OxdA and would donate a proton to the substrate in the aldoxime dehydration mechanism.
Konishi
Kazunobu
K
Institute of Applied Biochemistry, and Graduate School of Life and Environmental Sciences, The University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan.
Ishida
Kyoko
K
Oinuma
Ken-Ichi
K
Ohta
Takehiro
T
Hashimoto
Yoshiteru
Y
Higashibata
Hiroki
H
Kitagawa
Teizo
T
Kobayashi
Michihiko
M
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002244:Carbon / Q000737:chemistry* / Q000378:metabolism; D002248:Carbon Monoxide / Q000737:chemistry / Q000378:metabolism; D002942:Circular Dichroism; D006639:Histidine / Q000378:metabolism*; D006836:Hydro-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009584:Nitrogen / Q000737:chemistry* / Q000378:metabolism; D010091:Oximes / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013059:Spectrum Analysis, Raman; D014867:Water / Q000737:chemistry
D001426:Bacterial Proteins; D010091:Oximes; D011994:Recombinant Proteins; D014867:Water; D006639:Histidine; D002244:Carbon; D002248:Carbon Monoxide; D006836:Hydro-Lyases; C412124:aldoxime dehydratase; C023728:acetaldehyde oxime; D009584:Nitrogen