The Fe-type nitrile hydratase from Comamonas testosteroni Ni1 does not require an activator accessory protein for expression in Escherichia coli.
We report herein the functional expression of an Fe-type nitrile hydratase (NHase) without the co-expression of an activator protein or the Escherichia coli chaperone proteins GroES/EL. Soluble protein was obtained when the α- and β-subunit genes of the Fe-type NHase Comamonas testosteroni Ni1 (CtNHase) were synthesized with optimized E. coli codon usage and co-expressed. As a control, the Fe-type NHase from Rhodococcus equi TG328-2 (ReNHase) was expressed with (ReNHase(+Act)) and without (ReNHase(-Act)) its activator protein, establishing that expression of a fully functional, metallated ReNHase enzyme requires the co-expression of its activator protein, similar to all other Fe-type NHase enzymes reported to date, whereas the CtNHase does not. The X-ray crystal structure of CtNHase was determined to 2.4Å resolution revealing an αβ heterodimer, similar to other Fe-type NHase enzymes, except for two important differences. First, two His residues reside in the CtNHase active site that are not observed in other Fe-type NHase enzymes and second, the active site Fe(III) ion resides at the bottom of a wide solvent exposed channel. The solvent exposed active site, along with the two active site histidine residues, are hypothesized to play a role in iron incorporation in the absence of an activator protein.
Department of Chemistry and Biochemistry, Loyola University Chicago, 1068 W. Sheridan Rd., Chicago, IL 60626, United States.
Misty L
ML
Kuhn
Salette
S
Martinez
Natalie
N
Gumataotao
Uwe
U
Bornscheuer
Dali
D
Liu
Richard C
RC
Holz
D020134:Catalytic Domain; D020604:Comamonas testosteroni / Q000201:enzymology*; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000096:biosynthesis; D006360:Heat-Shock Proteins / Q000096:biosynthesis; D006639:Histidine / Q000737:chemistry; D006836:Hydro-Lyases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D007501:Iron / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D016929:Rhodococcus equi / Q000201:enzymology
D029968:Escherichia coli Proteins; C028715:GroE protein, E coli; D006360:Heat-Shock Proteins; D011994:Recombinant Proteins; D006639:Histidine; D007501:Iron; D006836:Hydro-Lyases; C048831:nitrile hydratase