NHases in Roseobacter

On the "Small Things Considered" blog (run by Prof Moselio Schaechter for the American Society for Microbiology), they have started an occasional series on interesting taxonomic groups of bacteria. The first that Prof Schaechter is giving is a pen picture of Roseobacter.

This group of bacteria are marine in origin, “make up 25% of the bacterial biomass in some coastal marine waters from the tropics to the poles” and have quite a lot of diversity in their microbiology so it seemed natural to see how much diversity there is in the recorded genomes for Roseobacter.

I did a search for nitrile hydratase alpha chains in organisms explicitly labelled as Roseobacter, and there are 7 which have RefSeq levels of quality in the NCBI database. There are two from Roseobacter litoralis Och 149, one from Roseobacter denitrificans OCh 114 and four from species various labelled sp. AZwk-3b, sp. CCS2, sp. SK209-2-6 and sp. MED193.  The two from Och 149 are very different but it can be seen from the COBALT alignment shown below that they are all cobalt containing NHases. According to a Clustal2.1 alignment, no two sequences are more than 88% similar (the second and third sequences are most similar, with the sixth/seventh pair next) with the average similarity being approximately 55%.

Diversity in Rhodococcus Sequences on NCBI

Downloading all the alpha chain amino acid sequences of nitrile hydratases of Rhodococcus origin from NCBI, you get over 100 sequences. By eliminating all the ones which are from PDB entries, you get 98 sequences. By using the usual amino acid sequence tag to identify which metal centre present, you get 68 iron-centred NHases and 24 cobalt-centred NHases, and six bits of rubbish. Of the iron centred ones, 61 have the VCSLC tag starting at position 109. None of the remaining have the metal binding region starting in the same place, and range from 96 to 149. There is much more of a spread with the cobalt centred sequences if you track the equivalent tag, as the histogram below shows.

Methods of immobilization of NHase, and a new one.

There is a new paper on the immobilization of nitrile hydratase to give greater stability. There are a few previous examples of this topic including:

·         Nitrile hydratase CLEAs: The immobilization and stabilization of an industrially important enzyme from Sander van Pelt, Sandrine Quignard, David Kubac, Dimitry Y. Sorokin, Fred van Rantwijk and Roger A. Sheldon in Green Chemistry in 2008. (DOI: 10.1039/b714258g)

·         Production of Acrylamide using Alginate-Immobilized E. coli Expressing Comamonas testosteroni 5-MGAM-4D Nitrile Hydratase from Lawrence J. Mersinger, Eugenia C. Hann, Frederick B. Cooling, John E. Gavagan, Arie Ben-Bassat, Shijun Wu, Kelly L. Petrillo, Mark S. Payne, and Robert DiCosimo in Advanced Synthesis and Catalysis in 2005. (DOI: 10.1002/adsc.200505039)

·         Biotransformation of nitriles by Rhodococcus equi A4 immobilized in LentiKats from David Kubáč, Alena Čejková, Jan Masák, Vladimír Jirků, Marielle Lemaire, Estelle Gallienne, Jean Bolte, Radek Stloukal, Ludmila Martínková in Journal of Molecular Catalysis B: Enzymatic in 2006. (doi:10.1016/j.molcatb.2006.01.004)

This one is Catalytic Properties of a Nitrile Hydratase Immobilized on Activated Chitosan by Yu. G. Maksimova, T. A. Rogozhnikova, G. V. Ovechkina, A. Yu. Maksimov, and V. A. Demakov in Applied Biochemistry and Microbiology (DOI: 10.1134/S0003683812030076).

They have used a nitrile hydratase isolated from a strain of Rhodococcus ruber gt1 and immobilized it on chitosan activated with 0.1% benzoquinone solution. They show that this immobilized enzyme can be used for 50 consecutive cycles of acrylonitrile transformation with activity holding up well.

They also found that their immobilized nitrile hydratases remain active at pH 3.0–4.0 which usefully extends its effective pH range.

Rotation

The quick and simple pdb2MGIF tool is available again at http://www.glycosciences.de/modeling/pdb2mgif/

Here is the nitrile hydratase from Comamonas tetosteroni Ni1 (4FM4) in two different rotating animations. I have cut the PDB file down so it only shows a single A/B dimer.

Overlaying the NHases in 4FM4 and 2QDY

The iron centred NHase which I have most experience of is the Rhodococcus erythropolis AJ270. I have overlayed it onto the structure for the Comomonas testosteroni Ni1, and it is very similar as you might expect both at the secondary structure level (4FM4 in red, 2QDY in blue and yellow) and as an alignment. (The original paper makes in-depth analysis with 1AHJ from Rhodococcus sp. r312.)

New publications- September 2012

There are two new publications:

• A review on stereoselectivity in nitrile hydratases by Anming Wang and co-workers in the African Journal of Microbiology Research (DOI: 10.5897/AJMR12.101). I am not sure it adds much to previous reviews. 
• A paper called “Biotransformation of benzonitrile herbicides via the nitrile hydratase–amidase pathway in rhodococci” in the Journal of Industrial Microbiology and Biotechnology with Ludmilla Martınkova as corresponding author (DOI: 10.1007/s10295-012-1184-z). It looks at the ability of Rhodococcus rhodochrous PA-34 and Rhodococcus erythropolis A4 to hydrate and hydrolyze chloroxynil, bromoxynil, iodoxynil and dichlorobenil. The NHase seemed to be able to turnover the herbicides more easily than the downstream amidases were able to hydrate the resulting amides.