A poster on Production of 2,6-difluorobenzamide using the NHase from Aurantimonas manganoxydans

Production of 2, 6- difluorobenzamide via whole-cell biocatalysis by nitrile hydratase from Aurantimonas manganoxydans

Lirong Yang

AGM Society for Industrial Microbiology & Biotechnology

Nitrile hydratases (NHases) are enzymes which catalyze the hydration of nitriles, converting them into their corresponding amides. Amides are an important intermediates for pharmaceutical and pesticide industry. For example, 2, 6 – difluorobenzamide is used for the synthesis of fluorinated benzoyl urea pesticide.

Four NHase genes from Aurantimonas manganoxydans ATCC BAA-1229, Klebsiella oxytoca KCTC 1686, Pseudomonas putida NRRL-18668, Comamonas testosteroni 5-MGAM-4D were cloned and functionally expressed in Escherichia coli BL21 (DE3). All of the recombinant NHases can catalyze the hydration of 2, 6-difluorobenzonitrile to produce 2, 6-difluorobenzamide. Among them, the NHase from Aurantimonas manganoxydans ATCC BAA-1229 showed the highest activity.

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.

 

A nitrile hydratase for cyanopyridines... (but it prefers aliphatic nitriles)

There has been a recent paper in Journal of Molecular Catalysis B on a nitrilase that converted cyanopyridines. This enzyme came from a strain of Pseudomonas putida. There is also a recent paper in the same journal entitled “Discovery of a new Fe-type nitrile hydratase efficiently hydrating aliphatic and aromatic nitriles by genome mining” by Xiaolin Peia, Lirong Yang, Gang Xu, Qiuyan Wang and Jianping Wu.which describes a nitrile hydratase, this time, from a Pseudomonas putida strain (F1) which they have shown to be able to turn over 3-cyanopyridine in a 1L fed batch reactor. Their activity data suggests it actually prefers aliphatic nitriles: acrylonitrile rates as 941 U/mg and valeronitrile as 535 U/mg as compared to 3-cyanopyridine at 26 U/mg. They describe how it was cloned into E. coli, needing the inclusion of an activator protein to get activity. They also include a nice phylogenetic tree showing the spread of known iron type NHases... plenty of examples in the Rhodococcus but spreading outwards into Pseudomonas.

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.

Looking inside for the active site of 3QXE

This is just a couple of rough pictures of 3QXE (the nitrile hydratase from Pseudomonas putida), once again from PyMol, showing a ribbon view of it with the active site cobalt-binding residues shown as CPK spheres, followed by a surface model with some of the active site just visible inside. I have been careful to keep it oriented similarly to the AJ270 nitrile hydratase in the previous post.

Du Pont and NHases

One of the interesting things about the paper "Evidence for Participation of Remote Residues...etc" by Ondrechen and Ringe is that the source of the recombinant NHase is Du Pont. Du Pont have quite a history of applying this class of enzyme to chemical problems. A search of the chemical or patent literature for the names "Robert Di Cosimo" (sometimes DiCosimo on Espacenet), "Mark S Payne" and "Robert Fallon" pulls up a range of uses of NHases.

For instance, WO 2006049618 which protects the NHase and amidase from Comamonas testosteroni 5-MGMA-4D from both authors, or the 1997 paper in Applied Microbiology and Biotechnology from Fallon, Stieglitz and Turner called "A Pseudomonas putida capable of stereoselective hydrolysis of nitriles" which describes work on the strain NRRL-18668.