A cobalt-containing eukaryotic nitrile hydratase.
Nitrile hydratase (NHase), an industrially important enzyme that catalyzes the hydration of nitriles to their corresponding amides, has only been characterized from prokaryotic microbes. The putative NHase from the eukaryotic unicellular choanoflagellate organism Monosiga brevicollis (MbNHase) was heterologously expressed in Escherichia coli. The resulting enzyme expressed as a single polypeptide with fused α- and β-subunits linked by a seventeen-histidine region. Size-exclusion chromatography indicated that MbNHase exists primarily as an (αβ)2homodimer in solution, analogous to the α2β2homotetramer architecture observed for prokaryotic NHases. The NHase enzyme contained its full complement of Co(III) and was fully functional without the co-expression of an activator protein or E. coli GroES/EL molecular chaperones. The homology model of MbNHase was developed identifying Cys400, Cys403, and Cys405 as active site ligands. The results presented here provide the first experimental data for a mature and active eukaryotic NHase with fused subunits. Since this new member of the NHase family is expressed from a single gene without the requirement of an activator protein, it represents an alternative biocatalyst for industrial syntheses of important amide compounds.
Department of Chemistry, Marquette University, PO Box 1881, Milwaukee, WI 53201-1881, United States; Department of Chemistry and Biochemistry, Loyola University Chicago, 1068 W Sheridan Rd., Chicago, IL 60660, United States.
Salette
S
Martinez
Department of Chemistry, Marquette University, PO Box 1881, Milwaukee, WI 53201-1881, United States.
Xinhang
X
Yang
Department of Physics, Marquette University, PO Box 1881, Milwaukee, WI 53201-1881, United States.
Brian
B
Bennett
Department of Chemistry, Marquette University, PO Box 1881, Milwaukee, WI 53201-1881, United States. Electronic address: richard.holz@marquette.edu.
Richard C
RC
Holz
D000595:Amino Acid Sequence; D056897:Choanoflagellata / Q000201:enzymology* / Q000235:genetics; D003035:Cobalt / Q000737:chemistry*; D018360:Crystallography, X-Ray; D006836:Hydro-Lyases / Q000737:chemistry* / Q000235:genetics; D008667:Metalloproteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D000072757:Protein Conformation, beta-Strand; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics; D013056:Spectrophotometry, Ultraviolet
D008667:Metalloproteins; D015800:Protozoan Proteins; D003035:Cobalt; D006836:Hydro-Lyases; C048831:nitrile hydratase
Cobalt; Enzyme kinetics; Hydrolysis; Nitrile hydratase