The Fe-type nitrile hydratase from Comamonas testosteroni Ni1 does not require an activator accessory protein for expression in Escherichia coli

Department of Chemistry and Biochemistry, Loyola University Chicago, 1068 W. Sheridan Rd., Chicago, IL 60626, United States.
Biochemical and Biophysical Research Communications (Impact Factor: 2.3). 06/2012; 424(3):365-70. DOI: 10.1016/j.bbrc.2012.06.036
Source: PubMed


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.

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    ABSTRACT: Microbial nitrile hydratases (NHases) are important industrial catalysts to produce valuable amides. However, only some NHase genes have been reported and studied at the molecular level. In this study, we developed a genome mining method to discover Fe-type NHases from GenBank. The putative NHase gene from Pseudomonas putida F1 was cloned and functionally expressed in Escherichia coli BL21 (DE3) by assisting of a putative activator gene adjacent to β-subunit region. Three recombinant plasmids containing NHase gene or the activator gene were designed and constructed. Maximal enzyme activity was obtained when the structural and activator genes were transcribed as one unit in plasmid pCDFDuet-1 at 18 °C. However, the expressed product did not show any NHase activity when the downstream activator gene was ignored, and the product completely existed in insoluble inclusion body. The activator gene might be involved in protein folding of the α- and β-subunits of NHase. In addition, the Fe-type NHase exhibited broad substrate specificity. The enzyme can efficiently hydrate aromatic nitriles, such as 3-cyanopyridine, 4-cyanopyridine, and benzonitrile, asides from aliphatic nitriles preferentially. Therefore, the recombinant NHase shows potential applications in some amides preparation.
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