NHase numbers to November 2012

Looking at the bare search term "nitrile hydratase" amongst protein sequences (and remember, most aren’t but it’s a rough measure), today gives me 4199 hits (+ 7% since August), of which 1605 (+ 8% since August) were RefSeq data.

No new PDB files have been deposited of nitrile hydratases in the last 3 months.

No nitrile hydratases in Acidobacteria

There is another interesting taxonomic post on All Things Considered called "A Whiff of Taxonomy: The Acidobacteria". They are a phylum of difficult to culture soil borne bacteria. I couldn't find one that contained a nitrile hydratase anything like the one in Rhodopseudomonas palustris CGA009. No evidence of the key metal binding sequence for either Co or Fe NHase at all. Some do appear to have nitrilase type proteins.

New thermophilic NHase sequence

I have the NCBI database set up to send me a weekly email digest of newly uploaded nitrile hydratase sequences. One of the ones which appeared this week is from the proteome of Mycobacterium hassiacum DSM 44199. This organism is one of the Mycobacterium which use humanity as its primary habitat, and this particular one was isolated from a urine sample collected in Germany in 1995. The literature reports that this organism is comfortable with temperatures up to 65 degrees C, and can deal with up to 5% salt, all of which might well offer typical "moderately thermophilic" properties to its nitrile hydratase. The alpha chain shows a "CTLCSC" sequence suggesting it is cobalt-centred, and BLASTing this sequence shows that 7 other Mycobacterium species are between 89-85% sequence similar, with non-Mycobacterium sequences being significantly different (starting below 55% similar). Interestingly most of the other Mycobacteria which are sources of these sequences are from environmental samples with much lower temperature ranges. I cant find any reference to a paper where a Mycobacterium has been exploited for its nitrile hydratase activity either in the Prasad and Bhalla review from 2010 or from a Google Scholar search. Perhaps here's one to start with.

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.