Metallochaperone function of the self‐subunit swapping chaperone involved in the maturation of subunit‐fused cobalt‐type nitrile hydratase

Abstract

The transition metal (iron or cobalt) is a mandatory part that constitutes the catalytic center of nitrile hydratase (NHase). The incorporation of the cobalt ion into cobalt‐containing NHase (Co‐NHase) was reported to depend on self‐subunit swapping and the activator of the Co‐NHase acts as a self‐subunit swapping chaperone for subunit exchange. Here we discovered that the activator acting as a metallochaperone transferred the cobalt ion into subunit‐fused Co‐NHase. We successfully isolated two activators, P14K and NhlE, which were the activators of NHases from Pseudomonas putida NRRL‐18668 and the activator of low‐molecular‐mass NHase from Rhodococcus rhodochrous J1, respectively. Cobalt content determination demonstrated that NhlE and P14K were two cobalt‐containing proteins. Substitution of the amino acids involved in the C‐terminus of the activators affected the activity of the two NHases, indicating that the potential cobalt‐binding sites might be located at the flexible C‐terminal region. The cobalt‐free NHases could be activated by either of the two activators, and both the two activators activated their cognate NHase more efficiently than did the noncognate ones. This study provided insights into the maturation of subunit‐fused NHases and confirmed the metallochaperone function of the self‐subunit swapping chaperone.

Proposed mechanism for post‐translational self‐modification of Co‐NHase based on Co2+ diffusion limitation

“…[5,26,27] However, all of these Co-NHases, made of independent or fused subunits, need to be coexpressed with activator proteins to incorporate Co 2+ into catalytic center of α-subunit for passive post-translational modification. [4,19] Studies had found that there was a high calculated energetic barrier (203 kcal mol -1 ) among the immature and mature NHases, and immature NHase needed an "external action" to overcome the energetic barrier and incorporate Co 2+ . [23] The highly flexible and positively charged C-domain of activator, which near the catalytic center, most likely performed an "external action" that overcame the energetic barrier and resulted in the mature Co-NHase.…”

“…[23] The highly flexible and positively charged C-domain of activator, which near the catalytic center, most likely performed an "external action" that overcame the energetic barrier and resulted in the mature Co-NHase. A large number of studies have shown that the activator had the metallochaperone function of the self-subunit swapping chaperone [19,21,28] and its Cterminal residue could specifically recognize metal ions. [29,30] In addition, when activator combined to the β-subunit, the catalytic center of α-subunit could be exposed to the solvent to facilitate the entry of Co 2+ .…”

“…Numerous studies have shown that the "activator proteins," such as P14K, NhlE, play a crucial role in assisting the activity expression of NHases. [10,19,20] These activators can bind iron or cobalt specifically as metallochaperones and transfer metal ions to the NHase catalysis center by self-subunit swapping to assist in the post-translation maturation of NHase. [21][22][23][24] Up to now, all the active expression of NHases could not be achieved without the assistance of activator, except that the Fe-NHase from Comamonas testosteroni Ni1 could be actively expressed in Escherichia coli without co-expression of activator.…”

Recent Advances and Promises in Nitrile Hydratase: From Mechanism to Industrial Applications

“…Previously, the Co-type activators were proposed to facilitate self-subunit swapping of NHase for enzyme maturation. However, by fusing the αand β-subunit as one single peptide, Zhou's group successfully identified the metallochaperone function of the Co-type activator (Xia et al, 2018b). By incubating apo-fused-NHase with Co-type activator, significant increase in the specific activity of apo-fused-NHases was observed, indicating that the activator can transfer cobalt ions to apo-NHases.…”

Efficient biodegradation of nitriles by a novel nitrile hydratase derived from Rhodococcus erythropolis CCM2595

“…NHases are classified into Co-type and Fe-type NHases according to the metal ions associated with the enzyme [1]. NHase catalyses the hydration of nitrile material to the corresponding amides, which are accessible from their respective nitrile precursors [2]. Nitrile precursors are widely used, especially in the manufacture of drugs, synthetic fibers and plastics, and often exhibit low-solubility in aqueous solutions [3,4].…”