Atrazine Chlorohydrolase from <i>Pseudomonas</i> Sp. Strain ADP Is a Metalloenzyme

Seffernick, Jennifer L.; McTavish, Hugh; Osborne, Jeffrey P.; Souza, M. L. De; Sadowsky, Michael J.; Wackett, Lawrence P.

doi:10.1021/bi020415s

Cited by 56 publications

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References 46 publications

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confidence: 92%

“…These structurally defined dehalogenases, which use water as a cosubstrate, are not metalloenzymes. In contrast, TrzN and AtzA each require a divalent metal ion to catalyze hydrolytic dehalogenation and thus differ mechanistically from other well studied halidohydrolases (4,11,13).TrzN and AtzA are both metalloenzymes of the amidohydrolase superfamily that contain zinc(II) and iron(II), respectively (11,13). A number of well studied amidohydrolases, such as adenosine deaminase (17) and cytosine deaminase (18), cata-* This work was supported by a grant from Sygenta Crop Protection (to L. P. W. and M. J. S.).…”

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confidence: 96%

“…TrzN and AtzA are both metalloenzymes of the amidohydrolase superfamily that contain zinc(II) and iron(II), respectively (11,13). A number of well studied amidohydrolases, such as adenosine deaminase (17) and cytosine deaminase (18), cata-* This work was supported by a grant from Sygenta Crop Protection (to L. P. W. and M. J. S.).…”

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

Seffernick

Reynolds

Fedorov

et al. 2010

Journal of Biological Chemistry

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Atrazine chlorohydrolase, TrzN (triazine hydrolase or atrazine chlorohydrolase 2), initiates bacterial metabolism of the herbicide atrazine by hydrolytic displacement of a chlorine substituent from the s-triazine ring. The present study describes crystal structures and reactivity of wild-type and active site mutant TrzN enzymes. The homodimer native enzyme structure, solved to 1.40 Å resolution, is a (␤␣) 8 barrel, characteristic of members of the amidohydrolase superfamily. TrzN uniquely positions threonine 325 in place of a conserved aspartate that ligates the metal in most mononuclear amidohydrolases superfamily members. The threonine side chain oxygen atom is 3.3 Å from the zinc atom and 2.6 Å from the oxygen atom of zinccoordinated water. Mutation of the threonine to a serine resulted in a 12-fold decrease in k cat /K m , largely due to k cat , whereas the T325D and T325E mutants had immeasurable activity. The structure and kinetics of TrzN are reminiscent of carbonic anhydrase, which uses a threonine to assist in positioning water for reaction with carbon dioxide. An isosteric substitution in the active site glutamate, E241Q, showed a large diminution in activity with ametryn, no detectable activity with atratone, and a 10-fold decrease with atrazine, when compared with wild-type TrzN. Activity with the E241Q mutant was nearly constant from pH 6.0 to 10.0, consistent with the loss of a proton-donating group. Structures for TrzN-E241Q were solved with bound ametryn and atratone to 1.93 and 1.64 Å resolution, respectively. Both structure and kinetic determinations suggest that the Glu 241 side chain provides a proton to N-1 of the s-triazine substrate to facilitate nucleophilic displacement at the adjacent C-2.There is substantial evidence that microbes rapidly evolve new enzymes to metabolize anthropogenic chemicals (1, 2). The s-triazine herbicides, such as atrazine, were first introduced into the environment 50 years ago and Ͼ2 billion pounds have been applied globally. s-Triazine compounds were initially found to be poorly biodegradable, but more rapid biodegradation is observed today (3). Many atrazine-degrading bacteria have now been isolated (4 -6). They invariably contain highly conserved genes encoding enzymes that hydrolytically displace substituents from the s-triazine ring carbon atoms to generate cyanuric acid (Fig. 1). The genes, trzN, atzA, atzB, and atzC, are found on plasmids and now are distributed globally (7, 8). These observations are consistent with the idea that a new metabolic pathway for atrazine catabolism may have evolved and spread in recent evolutionary times (9).The s-triazine herbicides, such as atrazine (2-chloro-4-isopropylamino-6-ethylamino-1,3,5-triazine) and ametryn (2-thiomethyl-4-isopropylamino-6-ethylamino-1,3,5-triazine), are metabolized readily by dedicated enzymes. The enzymes have been purified to homogeneity and shown to be inactive with structurally analogous pyrimidines and other closely related compounds (10 -13). Moreover, the enzymes are isolated from bacteria...

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

Seffernick

Reynolds

Fedorov

et al. 2010

Journal of Biological Chemistry

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“…Metal switching may also be a novel regulatory mechanism, whereby the activity and possibly stability of LpxC can be finely tuned by changes in intracellular metal ion concentrations. Furthermore, it is possible that many prokaryotic metal-dependent hydrolases similarly switch the active site metal between Fe(II) and Zn(II) in response to changes in cellular metal homeostasis, as suggested by the enzymes previously identified as zinc-or iron-dependent hydrolases (21,24,(57)(58)(59)(60). This newly proposed mechanism to regulate the activity of LpxC and possibly other metalloenzymes has important implications for understanding metalloenzyme homeostasis as well as future drug design for LpxC and other antibiotic targets.…”

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Active Site Metal Ion in UDP-3-O-((R)-3-Hydroxymyristoyl)-N-acetylglucosamine Deacetylase (LpxC) Switches between Fe(II) and Zn(II) Depending on Cellular Conditions*

Gattis

Hernick

Fierke

2010

Journal of Biological Chemistry

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UDP-3-O-((R)To analyze the native metal cofactor bound to LpxC, we developed a pulldown method to rapidly purify tagged LpxC under anaerobic conditions. The metal bound to LpxC purified from Escherichia coli grown in minimal medium is mainly Fe(II). However, the ratio of iron/ zinc bound to LpxC varies with the metal content of the medium. Furthermore, the iron/zinc ratio bound to native LpxC, determined by activity assays, has a similar dependence on the growth conditions. LpxC has significantly higher affinity for Zn(II) compared with Fe(II) with K D values of 60 ؎ 20 pM and 110 ؎ 40 nM, respectively. However, in vivo concentrations of readily exchangeable iron are significantly higher than zinc, suggesting that Fe(II) is the thermodynamically favored metal cofactor for LpxC under cellular conditions. These data indicate that LpxC expressed in E. coli grown in standard medium predominantly exists as the Fe(II)-enzyme. However, the metal cofactor in LpxC can switch between iron and zinc in response to perturbations in available metal ions. This alteration may be important for regulating the LpxC activity upon changes in environmental conditions and may be a general mechanism of regulating the activity of metalloenzymes.Gram-negative bacteria are important targets in the continuing fight against antibiotic-resistant infections. The development of new antibiotics to treat infections caused by resistant organisms is critically needed and will require novel targets. One potential source of targets in Gram-negative bacteria is the lipid A biosynthetic pathway (Fig. 1A) (1, 2), an essential building block of lipopolysaccharides (LPS) that make up the outer leaflet surrounding the cell wall in Gram-negative bacteria (2-4). In addition to being essential for cell viability, LPS are also referred to as endotoxins (2, 4, 5) and are responsible for immunogenic stimulation in septic shock that can lead to a number of deleterious effects, including severe hypotension, multiple organ dysfunction, and death (5). Consequently, inhibitors of lipid A biosynthesis have the potential to serve as both antibiotics and antiendotoxins (6). The UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) 3 catalyzes the committed and second overall step in lipid A biosynthesis, the hydrolysis of UDP-3-O-myristoyl-N-acetylglucosamine to UDP-3-O-myristoyl-glucosamine and acetate ( Fig. 1) (7-10). Consequently, inhibitors of LpxC are an active area of drug development (11)(12)(13)(14).LpxC was previously identified as a mononuclear Zn(II) metalloenzyme (Fig. 1B) (15) that also contains a weaker, inhibitory metal ion-binding site. This catalytic metal ion binds and polarizes an inner sphere water molecule that is activated by general base catalysis to serve as the nucleophile for hydrolysis of the acetyl substrate (Fig. 1C) (16, 17). However, recent evidence has shown that LpxC is more active with Fe(II) as its metal ion cofactor compared with Zn(II) (18). This increase in activity may correlate with a change in the ground stat...

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“…AtzA is in the same enzyme superfamily as cytosine deaminase and adenosine deaminase (Fig. 2B) and, like those enzymes, contains a mononuclear metal center, which is essential for catalytic activity (43). Adenosine deaminase catalyzes the hydrolytic deamination of adenosine and is, thus, a relatively common bacterial enzyme functioning in intermediary metabolism.…”

Section: Minireview: Evolution Of Microbial Enzymes 41260mentioning

confidence: 99%

Atrazine Chlorohydrolase from Pseudomonas Sp. Strain ADP Is a Metalloenzyme

Cited by 56 publications

References 46 publications

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

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

Active Site Metal Ion in UDP-3-O-((R)-3-Hydroxymyristoyl)-N-acetylglucosamine Deacetylase (LpxC) Switches between Fe(II) and Zn(II) Depending on Cellular Conditions*

Evolution of Enzymes for the Metabolism of New Chemical Inputs into the Environment

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