Maturation Mechanism of Nitrile Hydratase From Streptomyces canus CGMCC 13662 and Its Structural Character

Ling Guo,Xi Cheng,Yi-Jun Dai,Huo-Yong Jiang

doi:10.3389/fmicb.2020.01419

Abstract

Nitrile hydratases have received significant interest both in the large-scale industrial production of acrylamide and nicotinamide, and the remediation of environmental contamination with nitrile-containing pollutants. Almost all known nitrile hydratases include an α-subunit (AnhA) and β-subunit (AnhB), and a specific activator protein is crucial for their maturation and catalytic activity. Many studies exist on nitrile hydratase characteristics and applications, but few have reported their metal insertion and post-translational maturation mechanism. In this study, we investigated the cobalt insertion and maturation mechanism of nitrile hydratase from Streptomyces canus CGMCC 13662 (ScNHase) bearing three subunits (AnhD, AnhE, and AnhA). ScNHase subunits were purified, and the cobalt content and nitrile hydratase activity of the ScNHase subunits were detected. We discovered that cobalt could insert into the cobalt-free AnhA of ScNHase in the absence of activator protein under reduction agent DL-dithiothreitol (DTT) environment. AnhD not only performed the function of AnhB of NHase, but also acted as a metal ion chaperone and self-subunit swapping chaperone, while AnhE did not act as similar performance. A cobalt direct-insertion under reduction condition coordinated self-subunit swapping mechanism is responsible for ScNHase post-translational maturation. Molecular docking of ScNHase and substrates suggested that the substrate specificity of ScNHase was correlated with its structure. ScNHase had a weak hydrophobic interaction with IAN through protein–ligand interaction analysis and, therefore, had no affinity with indole-3-acetonitrile (IAN). The post-translational maturation mechanism and structure characteristics of ScNHase could help guide research on the environmental remediation of nitrile-containing waste contamination and three-subunit nitrile hydratase.

Highlights

Nitrile hydratase (NHase; EC 4.2.1.84) catalyzes the hydration of nitriles to give the corresponding amides (R–CN + H2O → R–CONH2) (Kobayashi et al, 1992; Yamada and Kobayashi, 1996; Kobayashi and Shimizu, 1998)
We found that cobalt can insert into all known nitrile hydratases include an α-subunit (AnhA) of ScNHase in the absence of activator protein under DTT added vitro environment, which is similar to the results of Pei et al (2013)
The cobalt contents in the related proteins verified that cobalt was able to insert into AnhA

Summary

Introduction

Nitrile hydratase (NHase; EC 4.2.1.84) catalyzes the hydration of nitriles to give the corresponding amides (R–CN + H2O → R–CONH2) (Kobayashi et al, 1992; Yamada and Kobayashi, 1996; Kobayashi and Shimizu, 1998). Most evidence has emphasized that the post-translational modification of cysteine into cysteine-sulfinic acid (αCys-SO2H) and cysteinesulfenic acid (αCys-SOH) is essential for catalytic efficiency (Nagashima et al, 1998; Murakami et al, 2000; Miyanaga et al, 2001; Hourai et al, 2003; Martinez et al, 2014). This metal-binding motif is known as the metal center of the metalloenzyme. Metallocenter biosynthesis mechanisms can be summarized as follows (Kuchar and Hausinger, 2004): (i) Reversible metal-ion binding; (ii) synergistic binding of metal with another component; (iii) metallochaperone delivery of metal ion or cofactor; (iv) post-translational modification to create a metal-binding site; (v) synthesis of metal-containing cofactors; (vi) requirement of an apoprotein-specific molecular chaperone; and (vii) metal incorporation coupled with electron transfer

Methods

Results

Discussion

Conclusion