Purification and Characterization of TrzF: Biuret Hydrolysis by Allophanate Hydrolase Supports Growth

ABSTRACT TrzF, the allophanate hydrolase from Enterobacter cloacae strain 99, was cloned, overexpressed in the presence of a chaperone protein, and purified to homogeneity. Native TrzF had a subunit molecular weight of 65,401 and a subunit stoichiometry of α2 and did not contain significant levels of metals. TrzF showed time-dependent inhibition by phenyl phosphorodiamidate and is a member of the amidase signature protein family. TrzF was highly active in the hydrolysis of allophanate but was not active with urea, despite having been previously considered a urea amidolyase. TrzF showed lower activity with malonamate, malonamide, and biuret. The allophanate hydrolase from Pseudomonas sp. strain ADP, AtzF, was also shown to hydrolyze biuret slowly. Since biuret and allophanate are consecutive metabolites in cyanuric acid metabolism, the low level of biuret hydrolase activity can have physiological significance. A recombinant Escherichia coli strain containing atzD, encoding cyanuric acid hydrolase that produces biuret, and atzF grew slowly on cyanuric acid as a source of nitrogen. The amount of growth produced was consistent with the liberation of 3 mol of ammonia from cyanuric acid. In vitro, TrzF was shown to hydrolyze biuret to liberate 3 mol of ammonia. The biuret hydrolyzing activity of TrzF might also be physiologically relevant in native strains. E. cloacae strain 99 grows on cyanuric acid with a significant accumulation of biuret.

Purification and Characterization of Allophanate Hydrolase (AtzF) from Pseudomonas sp. Strain ADP

ABSTRACT AtzF, allophanate hydrolase, is a recently discovered member of the amidase signature family that catalyzes the terminal reaction during metabolism of s-triazine ring compounds by bacteria. In the present study, the atzF gene from Pseudomonas sp. strain ADP was cloned and expressed as a His-tagged protein, and the protein was purified and characterized. AtzF had a deduced subunit molecular mass of 66,223, based on the gene sequence, and an estimated holoenzyme molecular mass of 260,000. The active protein did not contain detectable metals or organic cofactors. Purified AtzF hydrolyzed allophanate with a k cat/Km of 1.1 × 104 s−1 M−1, and 2 mol of ammonia was released per mol allophanate. The substrate range of AtzF was very narrow. Urea, biuret, hydroxyurea, methylcarbamate, and other structurally analogous compounds were not substrates for AtzF. Only malonamate, which strongly inhibited allophanate hydrolysis, was an alternative substrate, with a greatly reduced k cat/Km of 21 s−1 M−1. Data suggested that the AtzF catalytic cycle proceeds through a covalent substrate-enzyme intermediate. AtzF reacts with malonamate and hydroxylamine to generate malonohydroxamate, potentially derived from hydroxylamine capture of an enzyme-tethered acyl group. Three putative catalytically important residues, one lysine and two serines, were altered by site-directed mutagenesis, each with complete loss of enzyme activity. The identity of a putative serine nucleophile was probed using phenyl phosphorodiamidate that was shown to be a time-dependent inhibitor of AtzF. Inhibition was due to phosphoroamidation of Ser189 as shown by liquid chromatography/matrix-assisted laser desorption ionization mass spectrometry. The modified residue corresponds in sequence alignments to the nucleophilic serine previously identified in other members of the amidase signature family. Thus, AtzF affects the cleavage of three carbon-to-nitrogen bonds via a mechanism similar to that of enzymes catalyzing single-amide-bond cleavage reactions. AtzF orthologs appear to be widespread among bacteria.

Tests of nitrification and of urease inhibitors, when applied with either solid or aqueous urea, on grass grown on a light sandy soil

SummaryIn 1984 and 1985 a field experiment on a grass ley on a light sandy soil at Woburn Experimental Farm, Bedfordshire, tested injected aqueous urea and broadcast prilled urea, applied alone or with a nitrification or urease inhibitor. Aqueous urea, prilled urea and ‘Nitro-Chalk’ were applied as a single 375 kg N/ha dressing, and prilled urea and ‘Nitro-Chalk’ also as three 125 kg N/ha dressings. The nitrification inhibitor nitrapyrin or a mixture of sodium trithiocarbonate (STC) plus potassium ethyl xanthate (KEtX) was injected with aqueous urea. The nitrification inhibitor dicyandiamide (DCD) or the urease inhibitor phenyl-phosphorodiamidate (PPDA) was broadcast with prilled urea.The nitrification inhibitors significantly retarded nitrification of both aqueous and prilled urea. PPDA reduced ammonia volatilization from 375 kg N/ha broadcast as urea, and hence losses to the atmosphere, which otherwise ranged from 13 to 33 kg N/ha.Nitrapyrin or STC and KEtX increased yield and nitrogen uptake in both years when urea was injected in January. Nitrapyrin also increased yield and nitrogen uptake in 1985, but not in 1984, when urea was injected in March, whereas the STC and KEtX mixture was then either detrimental or ineffective. DCD increased yield and nitrogen uptake from a single dressing of broadoast urea only in 1985. PPDA increased yield and nitrogen uptake from a single broadcast dressing of urea in both years, but had little effect when applied with divided dressings.In 1984 a divided broadcast dressing of ‘Nitro-Chalk’ gave the largest yield and nitrogen uptake, but in 1985 aqueous urea injected with nitrapyrin in January or without or with a nitrification inhibitor in Maroh and prilled urea broadcast as a divided dressing all gave a larger yield. Similarly, a single application was generally more effective as ‘Nitro-Chalk’ in 1984, but as urea in 1985.