A switch in a substrate tunnel for directing regioselectivity of nitrile hydratases towards α,ω-dinitriles

A switch in a substrate tunnel for directing regioselectivity of nitrile hydratases towards α,ω-dinitriles

Zhongyi Cheng, Wenjing Cui, Zhongmei Liu, Li Zhou, Min Wang, Michihiko Kobayashi and Zhemin Zhou 

The β37 residue of nitrile hydratase (NHase) from Pseudomonas putida and NHase from Comamonas testosteroni played a critical role in directing enzyme regioselectivity. Amino acid substitution in this site modulated or even inverted enzyme regioselectivity towards aliphatic α,ω-dinitriles.

Cartoon model of the substrate access tunnel of (a) wild-type PpNHase and its (b) L37F and (c) L37Y variants, and (d) wild-type CtNHase and its (e) F37L and (f) F37P variants. The protein structures of PpNHase and CtNHase are shown as the grey cartoon. The β37 residues of NHases are shown as blue sticks. The purple balls and sticks represent the catalytic site of NHase. The bottleneck-forming amino acids are shown as red sticks. The tunnels are shown as green spheres, and the tunnel bottlenecks are coloured in yellow. All the figures share the same size proportion.

Enhancement of NHase stability with self assembling peptides

There is an in-press paper available online entitled “Enhancement ofthermo-stability and product tolerance of Pseudomonasputida nitrile hydratase by fusing with self-assembling peptide” in the Journal of Bioscience and Bioengineering by Zhemin Zhou and co-workers. They describe how they have used some self-assembling peptide based tags appended to the beta subunit to enhance thermal stability and substrate tolerance.

How does the nitrile hydratase activator protein work?

This is a question which is still up for debate. It would appear to be involved with incorporation of the cobalt ion in those NHases which are cobalt-centred. In a newly accepted manuscript of FEMS Microbiology Letters entitled “The effect of flexibility and positive charge of the C‐terminal domain on the activator P14K function for nitrile hydratase in Pseudomonas putida” by Zhemin Zhou and co-workers, mutants of the relevant proteins were modelled and made, and then tested in the hydration of 3-cyanopyridine.

 

New nitrile hydratase papers

Enzyme–Substrate Binding Landscapes in the Process of Nitrile Biodegradation Mediated by Nitrile Hydratase and Amidase from Yu Zhang, Zhuotong Zeng, Guangming Zeng, Xuanming Liu, Ming Chen, Lifeng Liu, Zhifeng Liu & Gengxin Xie in Applied Biochemistry and Biotechnology describes molecular modelling experiments using the crystal structures 2QDY (AJ270 Fe based NHase) and 1IRE (Pseudonocardia thermophila Co based NHase).This is a docking study for these two enzymes and a downstream amidase. Available at DOI 10.1007/s12010-013-0276-1. Shown below is Fig1b which illustrates a binding mode between the P. thermophila Co based NHase and 3-cyanopyridine.

Strategy for successful expression of the Pseudomonas putida nitrile hydratase activator P14K in Escherichia coli by Yi Liu, Wenjing Cui , Yueqin Fang, Yuechun Yu, Youtian Cui, Yuanyuan Xia, Michihiko Kobayashi and Zhemin Zhou adds to debate around the activator which is used for the maturation of the NHase with inclusion of the metal centre  (they say an activator is always needed, but is that true?). This paper is found in BMC Biotechnology at DOI:10.1186/1472-6750-13-48 and describes a methodology to ensure that the P14K activator is expressed successfully and in a more stable form.

Self-Subunit Swapping Occurs in Another Gene Type of Cobalt Nitrile Hydratase

Liu Y, Cui W, Xia Y, Cui Y, Kobayashi M, et al. (2012) Self-Subunit Swapping Occurs in Another Gene Type of Cobalt Nitrile Hydratase. PLoS ONE 7(11): e50829. doi:10.1371/journal.pone.0050829.