Assisting soluble expression of recombinant and active Co-centred NHase

Chaperones-assisted soluble expression and maturation of recombinant Co-type nitrile hydratase in Escherichia coli to avoid the need for a low induction temperature

Xiaolin Pei, Qiuyan Wang, Lijun Meng, Jing Li, Zhengfen Yang, Xiaopu Yin, Lirong Yang, Shaoyun Chen and Jianping Wu

Journal of Biotechnology, Volume 203, 10 June 2015, Pages 9–16

doi:10.1016/j.jbiotec.2015.03.004

Nitrile hydratase (NHase) is an important industrial enzyme that biosynthesizes high-value amides. However, most of NHases expressed in Escherichia coli easily aggregate to inactive inclusion bodies unless the induction temperature is reduced to approximately 20 °C. The NHase from Aurantimonas manganoxydans has been functionally expressed in E. coli, and exhibits considerable potential for the production of nicotinamide in industrial application. In this study, the effects of chaperones including GroEL/ES, Dnak/J-GrpE and trigger factor on the expression of the recombinant Co-type NHase were investigated. The results indicate that three chaperones can significantly promote the active expression of the recombinant NHase at 30 °C. The total NHase activities reached to 263 and 155 U/ml in shake flasks when the NHase was co-expressed with GroEL/ES and DnaK/J-GrpE, which were 52- and 31-fold higher than the observed activities without chaperones, respectively. This increase is possibly due to the soluble expression of the recombinant NHase assisted by molecular chaperones. Furthermore, GroEL/ES and DnaK/J-GrpE were determined to promote the maturation of the Co-type NHase in E. coli under the absence of the parental activator gene. These knowledge regarding the chaperones effect on the NHase expression are useful for understanding the biosynthesis of Co-type NHase.

Clues towards the role of amide carbonyl groups in the active site of a cobalt centred NHase

Role of the Amide Carbonyl Groups in the Nitrile Hydratase Active Site for Nitrile Coordination Using Co(III) Complex with N2S3-type Ligand

Takuma Yano,  Tomohiro Ikeda, Tomonori Shibayama,  Tomohiko Inomata, Yasuhiro Funahashi,2 Tomohiro Ozawa,* and Hideki Masuda*

Chemistry Letters Vol. 44 (2015) No. 6 P 761-763

The role of the amide carbonyl oxygens in the nitrile hydratase (NHase) active site for the nitrile coordination to the metal center was studied using a distorted square-pyramidal N2S3-type Co(III) complex at room temperature in the presence of a folded-sheet mesoporous material (FSM) or sodium cation.

Characterization of the NHase from Ensifer meliloti CGMCC 7333

Characterization of a versatile nitrile hydratase of the neonicotinoid thiacloprid-degrading bacterium Ensifer meliloti CGMCC 7333

Shi-Lei Sun, Tian-Qi Lu, Wen-Long Yang, Jing-Jing Guo, Xue Rui, Shi-Yun Mao, Ling-Yan Zhou and Yi-Jun Dai - RSC Advances, 2016

The nitrogen-fixing bacterium Ensifer meliloti CGMCC 7333 and its nitrile hydratase (NHase) degrade the neonicotinoid insecticides, thiacloprid (THI) and acetamiprid (ACE), to their corresponding amide metabolites. The NHase gene cluster is composed of α-subunit and β-subunit genes and a hypothetical protein gene. The functionality of the hypothetical protein downstream of the NHase coding genes and the characteristics of CGMCC 7333 NHase were explored in this study. Co-expression of the hypothetical protein coding gene with NHase (α- and β-subunit genes) in Escherichia coli Rosetta enhanced NHase hydration of THI and ACE two- and four-fold, respectively, and also significantly improved NHase solubility compared with the absence of the hypothetical protein coding gene. The NHase displayed an optimal reaction temperature of 50 °C for THI hydration and was unstable when the incubation temperature exceeded 40 °C. The optimum reaction pH was 7.0 and the NHase activity was stable in the pH range of 6 to 9. The enzyme activity for THI hydration was slightly inhibited by copper, zinc, and iron, and decreased by 68.6%, 75.7%, and 70.3% when 2% ethanol, ethyl acetate, and acetone were added to the reaction mixture, respectively, whereas dichloromethane and trichloromethane had no effect. The K_m and k_cat values of CGMCC 7333 NHase for THI hydration were 12.39 mmol L⁻¹ and 131.36 s⁻¹, respectively. Substrate specificity analysis indicated that CGMCC 7333 NHase also transformed 3-cyanopyridine, benzonitrile, and indole-3-acetonitrile to the corresponding amide products, with maximum specific activities of 652.52, 255.32, and 263.93 U mg⁻¹ protein, respectively.

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.

 

E. coli expression of active nitrile hydratase from Aurantimonas manganoxydans needs cobalt ions

This paper in Biotechnology Letters from Pei and co-workers gives details of a cobalt centred nitrile hydratase from Aurantimonas manganoxydans, and the importance of getting the concentration right for good level of expression and activity. (DOI 10.1007/s10529-013-1215-5)

Sequential Oxidations of Thiolates and the Cobalt Metallocenter in a Synthetic Metallopeptide: Implications for the Biosynthesis of Nitrile Hydratase

This is a paper in Inorganic Chemistry from the group of Anne K Jones looking at the use of a heptapeptide they call SODA (ACDLPCG) to model the oxidation process for the thiols and metal in the active site of cobalt centre NHases (DOI: 10.1021/ic400171z). First to oxidize is one of the thiolates to sulfinate, then the cobalt is oxidized to triply charged cobalt. Further sulphur oxidation is not observed and the complex is catalytically inactive. This complex does not have an axial thiolate ligand which may explain these last two obervations.

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.

S-M bond lengths

I was wondering what a "normal" bond length might be for a thiol to iron or cobalt might be, and then what might happen to that bond length might do if that bond was oxidized once and then twice. The best way to do this is to look at the small molecule crystallographic data so I got my friend Ross to comb through the usual database to find examples of the various bonding motifs.

It turns out that there are no examples of 1b in the CCD, and precious few of 1c (only 4), 2b (only 5) and 2c (a more respectable 15). There are loads of examples of 1d (actually 286) and 2d (102).
Anyway, there isnt much of a difference in average bond lengths between these types: 1c- 2.221 angstroms, 1d- 2.276 angstroms, 2b- 2.225 angstroms, 2c- 2.208 angstroms and 2d- 2.252 angstroms.
Here are a few histograms showing the distributions that lie behind these means.

A predicted nitrile hydratase from a psychrophilic bacterium

I have been scanning the sequences, and by chance I came across the fact that there is a predicted nitrile hydratase from a bacterium called Octadecabacter antarcticus strain 307 which is a marine organism found at the poles. From the looks of the primary amino acid sequence of the alpha subunit (gb|EDY76981.1|), it is a cobalt-centred NHase.

Cobalt binding in 3QXE

Above is an excerpt from Figure 6C in "Evidence for Participation of Remote Residues...etc by Ondrechen and Ringe which very nicely shows how the cobalt ion is bound into a nitrile hydratase.  There are three sulphur based points of attachment- aC112, aC115 and aC117, the latter two as sulfinic acids and two amide nitrogen points of attachment, aS116 and aC117. It is really clearly shown in this view how the arginine bR52 preorganizes the sulfinic cysteines and amide nitrogens into a planar SSNN arrangement.

Common features of cobalt and iron based NHases

With a couple of local colleagues, I have a paper in press with the International Journal of Data Mining and Bioinformatics. It is the eventual result of Joe, a mathematician asking if I had any high dimensional data sets that needed interrogating. In terms of this blog, it has a section looking at what are the main primary sequence features (using a set of one hundred NHases) which are important in distinguishing cobalt centred NHases from iron centred ones. Thanks to Marcus for all the 3D structure analysis, and spotting the hinge/loop differences first suggested as relevant to differential stability by Miyanaga in 2001

http://northumbria.academia.edu/JoeFaith/Papers/320396/Predicting_Functional_Residues_of_Protein_Sequence_Alignments_as_a_Feature_Selection_Task

First Post

The point of this blog is to follow what is going with one particular class of enzyme, nitrile hydratase. I may stray the odd time into posting about nitrilases or amidase but my research interest is more on NHases.

First up, let me give a couple of references to NHase papers where I am corresponding author:

Biotransformation of nitriles using the solvent-tolerant nitrile hydratase from Rhodopseudomonas palustris CGA009- Black, Gregson, McPake, Perry and Zhang in Tetrahedron Letters (2010) doi:10.1016/j.tetlet.2009.12.094

Probing the Enantioselectivity of a Diverse Group of Purified Cobalt-Centred Nitrile Hydratases- van Pelt, Zhang, Otten, Holt, Sorokin, Rantwijk, Black, Perry and Sheldon (accepted manuscript, 2011) DOI: 10.1039/C0OB01067G

The recombinant enzymes mentioned in these papers are available for purchase through a few companies, namely Prozomix, Almac and CLEA Technologies.

If you are a UK/EU based academic, and interested in working with our enzymes, feel free to contact me.