Evidence for the participation of an extra α-helix at β-subunit surface in the thermal stability of Co-type nitrile hydratase
- 255 Downloads
Nitrile hydratase (NHase) has attracted considerable attention since it can efficiently catalyze the hydration of nitriles to valuable amides. However, the poor stability of NHase is one of the main drawbacks in the industrial application. In this study, we compared the structural difference between Fe-type and Co-type NHase and found that an extra α helix existed at the β-subunit surface of Co-type NHase (defined as the β-6th helix). Then, the effects of the β-6th helix were investigated on the thermal stability and the catalytic kinetics of a Co-type NHase from Aurantimonas manganoxydans ATCC BAA-1229 (NHase1229). When the β-6th helix was deleted or disrupted, the thermal stability of NHase1229 was reduced to 17.6 and 12.9% of that of wild NHase1229, respectively. Thus, the β-6th helix is important for the thermal stability of Co-type NHase. Based on the structural characteristics of Co-type NHase, the β-6th helix may be interacted with another helix at the α-subunit (defined as the α-2nd helix) by hydrophobic network just as a “magnetic suction buckle” on the enzyme surface to stabilize the binding of α- and β-subunits. The β-6th helix is located at the mouth of the substrate and product tunnel, so it plays crucial roles in catalytic process. Furthermore, the β-6th helix in NHase1229 was swapped with a thermophilic NHase fragment from Pseudonocardia thermophila JCM3095 (NHase1229-Swap). The thermal stability of NHase1229-Swap was significantly improved, and the half-life was approximately 2.4-fold at 40 °C than that of the wild NHase1229. The knowledge is useful for improving the stability of NHases by restriction fragment swapping.
KeywordsNitrile hydratase Thermal stability Thermal-sensitive region Fragment swapping Chimeric enzyme
This study was funded by the Natural Science Foundation of Zhejiang Province (No. LY15B060011, LY17C050001, LY18B060009, Q12B06007), the China Postdoctoral Science Foundation Funded Project (No. 2016M601966), the National Natural Science Foundation of China (Nos. 21576062, 21206024), and the Hangzhou Science and Technology Project (Nos. 20130533B17, 20151232I34).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
- Hourai S, Ishii T, Miki M, Takashima Y, Mitsuda S, Yanagi K (2005) Cloning, purification, crystallization and preliminary X-ray diffraction analysis of nitrile hydratase from the thermophilic Bacillus smithii SC-J05-1. Acta Cryst F61:974–977. https://doi.org/10.1107/S1744309105030939 CrossRefGoogle Scholar
- Nagasawa T, Nanba H, Ryuno K, Takeuchi K, Yamada H (1987) Nitrile hydratase of Pseudomonas chlororaphis B23. Purification and characterization. Eur J Biochem 162:691–698. https://doi.org/10.1111/j.1432-1033.1987.tb10692.x CrossRefPubMedGoogle Scholar
- Petrillo KL, Wu S, Hann EC, Cooling FB, Ben-Bassat A, Gavagan JE, DiCosimo R, Payne MS (2005) Over-expression in Escherichia coli of a thermally stable and regio-selective nitrile hydratase from Comamonas testosteroni 5-MGAM-4D. Appl Microbiol Biot 67:664–670. https://doi.org/10.1007/s00253-004-1842-9 CrossRefGoogle Scholar
- Rzeznicka K, Schätzle S, Böttcher D, Klein J, Bornscheuer UT (2010) Cloning and functional expression of a nitrile hydratase (NHase) from Rhodococcus equi TG328-2 in Escherichia coli, its purification and biochemical characterization. Appl Microbiol Biot 85:1417–1425. https://doi.org/10.1007/s00253-009-2153-y CrossRefGoogle Scholar
- Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, Söding J, Thompson JD, Higgins DG (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7:539. https://doi.org/10.1038/msb.2011.75 CrossRefPubMedPubMedCentralGoogle Scholar
- Song L, Wang M, Shi J, Xue Z, Wang MX, Qian S (2007) High resolution X-ray molecular structure of the nitrile hydratase from Rhodococcus erythropolis AJ270 reveals posttranslational oxidation of two cysteines into sulfinic acids and a novel biocatalytic nitrile hydration mechanism. Biochem Bioph Res Co 362:319–324. https://doi.org/10.1016/j.bbrc.2007.07.184 CrossRefGoogle Scholar