Identification and functional analysis of the activator gene involved in the biosynthesis of Co-type nitrile hydratase from Aurantimonas manganoxydans

Abstract

Nitrile hydratase (NHase) has attracted considerable attention to synthesize valuable amides. Here, the gene cluster of Co-type NHase from Aurantimonas manganoxydans ATCC BAA-1229 (NHase1229) was identified through Edman degradation and recombinant expression, including α-subunit, β-subunit, and activator. The activator gene contained 408 bases, which was essential for the biosynthesis of NHase1229 in Escherichia coli. The activator gene can be substituted by Co-type activator from Pseudonocardia thermophila JCM3095 and Pseudomonas putida 5B to assist in the functional expression of NHase1229, not by that of Fe-type NHase from Pseudomonas putida F1. Multi-sequence alignment showed that the activator contained conserved amino acid residues (Trp34, Trp53, Phe 85, Gln35, Glu39, and Glu90), but these residues had no significant effect on activator function. Histidine residues (His112, His120, and His134) at the C-terminus only partially participated in the activator function. Furthermore, the successive α-helices in domain TIGR03889 were identified to be critical for the activator function. The integration efficiency of cobalt into the active site was decreased with the interruption of α-helices. Therefore, Co-type and Fe-type activators have specific functions for the integration of different metal ions, and four conserved α-helices in Co-type activator affect the integration of cobalt. These knowledge is useful for understanding the biosynthesis of Co-type NHase.