Substrate Specificity and Colorimetric Assay for Recombinant TrzN Derived from Arthrobacter aurescens TC1

Abstract

The TrzN protein, which is involved in s-triazine herbicide catabolism by Arthrobacter aurescens TC1, was cloned and expressed in Escherichia coli as a His-tagged protein. The recombinant protein was purified via nickel column chromatography. The purified TrzN protein was tested with 31 s-triazine and pyrimidine ring compounds; 22 of the tested compounds were substrates. TrzN showed high activity with sulfur-substituted s-triazines and the highest activity with ametryn sulfoxide. Hydrolysis of ametryn sulfoxide by TrzN, both in vitro and in vivo, yielded a product(s) that reacted with 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) to generate a diagnostic blue product. Atrazine chlorohydrolase, AtzA, did not hydrolyze ametryn sulfoxide, and no color was formed by amending those enzyme incubations with NBD-Cl. TrzN and AtzA could also be distinguished by reaction with ametryn. TrzN, but not AtzA, hydrolyzed ametryn to methylmercaptan. Methylmercaptan reacted with NBD-Cl to produce a diagnostic yellow product having an absorption maximum at 420 nm. The yellow color with ametryn was shown to selectively demonstrate the presence of TrzN, but not AtzA or other enzymes, in whole microbial cells. The present study was the first to purify an active TrzN protein in recombinant form and develop a colorimetric test for determining TrzN activity, and it significantly extends the known substrate range for TrzN.

X-ray Structure and Mutational Analysis of the Atrazine Chlorohydrolase TrzN

“…There is, however, a channel occupied by a set of three waters (57.S, 102.S, and 112.S), which has the first water residing 4.6 Å from the methylene carbon of the ethyl group, which could potentially house a longer side chain. This may be how TrzN catalyzes reactions with symmetrical substrates containing longer and more branched side chains at both N-alkyl positions, albeit at reduced rates (10). The N-isopropyl binding pocket is slightly larger.…”

“…A number of well studied amidohydrolases, such as adenosine deaminase (17) and cytosine deaminase (18), cata-lyze hydrolytic deamination from diazine ring systems, which is somewhat comparable to hydrolytic dechlorination from an s-triazine ring substrate. Yet, AtzA has a high degree of substrate specificity, catalyzing only dehalogenation reactions (10). In contrast, TrzN has a broader substrate specificity and displaces thiomethyl and methoxy groups from the related s-triazine herbicides ametryn and atratone (2-methoxy-4-isopropylamino-6-ethylamino-1,3,5-triazine), respectively.…”

Evolution of Catabolic Pathways: Genomic Insights into Microbial s -Triazine Metabolism

“…TrzN was first investigated by Topp and coworkers (75) and was shown to have a significantly broader substrate range than AtzA. A more recent study expanded the known substrate range of TrzN to include other herbicides and analogs of commercial herbicides (66). At least eight distinct leaving groups are hydrolytically displaced from carbon 2 of the s-triazine ring, and TrzN tolerates a wide range of different N-alkyl groups at the 4 and 6 carbon atoms.…”

Hydroxyatrazine N -Ethylaminohydrolase (AtzB): an Amidohydrolase Superfamily Enzyme Catalyzing Deamination and Dechlorination

“…The work described here outlines a novel approach for cloning the atzB gene that allowed for further enzyme characterization, including a detailed study on substrate specificity. 2-isopropylamino-4-ethylamino-1,3,5-triazine, and 2-mercapto-4-isopropylamino-6-ethylamino-1,3,5-triazine were synthesized as previously described (26,33). Compounds synthesized for this study were analyzed for purity by gas chromatography-mass spectrometry (MS) using an HP 6890/5973 instrument (Hewlett-Packard, San Fernando, CA).…”