PMID:16885454

Citation	Shapir, N, Pedersen, C, Gil, O, Strong, L, Seffernick, J, Sadowsky, MJ and Wackett, LP (2006) TrzN from Arthrobacter aurescens TC1 Is a zinc amidohydrolase. J. Bacteriol. 188:5859-64
Abstract	TrzN, the broad-specificity triazine hydrolase from Arthrobacter and Nocardioides spp., is reportedly in the amidohydrolase superfamily of metalloenzymes, but previous studies suggested that a metal was not required for activity. To help resolve that conundrum, a double chaperone expression system was used to produce multimilligram quantities of functionally folded, recombinant TrzN. The TrzN obtained from Escherichia coli (trzN) cells cultured with increasing zinc in the growth medium showed corresponding increases in specific activity, and enzyme obtained from cells grown with 500 muM zinc showed maximum activity. Recombinant TrzN contained 1 mole of Zn per mole of TrzN subunit. Maximally active TrzN was not affected by supplementation with most metals nor by EDTA, consistent with previous observations (E. Topp, W. M. Mulbry, H. Zhu, S. M. Nour, and D. Cuppels, Appl. Environ. Microbiol. 66:3134-3141, 2000) which had led to the conclusion that TrzN is not a metalloenzyme. Fully active native TrzN showed a loss of greater than 90% of enzyme activity and bound zinc when treated with the metal chelator 8-hydroxyquinoline-5-sulfonic acid. While exogenously added zinc or cobalt restored activity to metal-depleted TrzN, cobalt supported lower activity than did zinc. Iron, manganese, nickel, and copper did not support TrzN activity. Both Zn- and Co-TrzN showed different relative activities with different s-triazine substrates. Co-TrzN showed a visible absorption spectrum characteristic of other members of the amidohydrolase superfamily replaced with cobalt.
Links	PubMed PMC1540083 Online version:10.1128/JB.00517-06
Keywords	Amidohydrolases/chemistry; Amidohydrolases/metabolism; Amino Acid Sequence; Arthrobacter/enzymology; Arthrobacter/genetics; Cobalt/chemistry; Kinetics; Zinc/chemistry
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
ARTAU:Q6SJY7		GO:0004040: amidase activity	ECO:0000314:		F		Figure 5 demonstrates the activity of Zn(II) and Co(II) trzN with selected s-triazines as substrates. Figure 4 shows the effect of metal addition on ametryn hydrolysis activity of native TrzN purified from recombinant cells grown on 500M zinc and metal-depleted TrzN.	complete CACAO 7543
ARTAU:Q6SJY7	enables	GO:0004040: amidase activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete

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