Catalytic Improvement and Evolution of Atrazine Chlorohydrolase

Scott, Colin; Jackson, Colin J.; Coppin, Chris; Mourant, Roslyn; Hilton, Margaret E.; Sutherland, Tara D.; Russell, Robyn J.; Oakeshott, John G.

doi:10.1128/aem.02634-08

Cited by 58 publications

(45 citation statements)

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“…To improve the yield of its soluble form, we performed laboratory evolution, using a method that we have previously used with related metallohydrolases from Pseudomonas sp. strain ADP (AtzA [atrazine chlorohydrolase]) and Agrobacterium radiobacter (phosphotriesterase) (5,57). This involved screening libraries of random variants using an agar plate-based activity screen in which substrates were included in the agar gel at concentrations in excess of the solubility limit, resulting in opaque plates.…”

Section: Resultsmentioning

confidence: 99%

“…These metal ions can play structural or catalytic roles, and their incorporation into apoenzyme folding intermediates is an important step in the overall folding pathway (2). One of the most diverse superfamilies of enzymes, which includes a large number of microbial enzymes that are capable of catalyzing the hydrolysis of toxic synthetic compounds (3)(4)(5), is the metal ion-dependent amidohydrolase superfamily (6,7). Unfortunately, many of these proteins form insoluble aggregates or are produced only at very low levels when overexpressed in recombinant systems.…”

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confidence: 99%

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300-Fold Increase in Production of the Zn ²⁺ -Dependent Dechlorinase TrzN in Soluble Form via Apoenzyme Stabilization

Jackson

Coppin

Carr

et al. 2014

Appl Environ Microbiol

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Microbial metalloenzymes constitute a large library of biocatalysts, a number of which have already been shown to catalyze the breakdown of toxic chemicals or industrially relevant chemical transformations. However, while there is considerable interest in harnessing these catalysts for biotechnology, for many of the enzymes, their large-scale production in active, soluble form in recombinant systems is a significant barrier to their use. In this work, we demonstrate that as few as three mutations can result in a 300-fold increase in the expression of soluble TrzN, an enzyme from Arthrobacter aurescens with environmental applications that catalyzes the hydrolysis of triazine herbicides, in Escherichia coli. Using a combination of X-ray crystallography, kinetic analysis, and computational simulation, we show that the majority of the improvement in expression is due to stabilization of the apoenzyme rather than the metal ion-bound holoenzyme. This provides a structural and mechanistic explanation for the observation that many compensatory mutations can increase levels of soluble-protein production without increasing the stability of the final, active form of the enzyme. This study provides a molecular understanding of the importance of the stability of metal ion free states to the accumulation of soluble protein and shows that differences between apoenzyme and holoenzyme structures can result in mutations affecting the stability of either state differently.A pproximately half of all known proteins contain a metal ion cofactor in their mature form (1). These metal ions can play structural or catalytic roles, and their incorporation into apoenzyme folding intermediates is an important step in the overall folding pathway (2). One of the most diverse superfamilies of enzymes, which includes a large number of microbial enzymes that are capable of catalyzing the hydrolysis of toxic synthetic compounds (3-5), is the metal ion-dependent amidohydrolase superfamily (6, 7). Unfortunately, many of these proteins form insoluble aggregates or are produced only at very low levels when overexpressed in recombinant systems.There is a need to improve heterologous protein expression in order to enable us to characterize proteins in the laboratory or to produce proteins on a large scale for industrial or medical purposes. Heterologous overexpression of proteins is often less than ideal because of protein aggregation or degradation (8). This can arise due to differences between the native folding situation and that which occurs when expression levels are much higher and in a different host, such as a lack of suitable folding chaperones, different pH, high local concentration of the protein, or the absence of a cofactor, which is particularly important for metalloproteins. The technique of laboratory evolution has great potential for improving soluble-protein production and involves subjecting the protein to random mutagenesis and recombination, followed by screening of the many variants that are produced for enhanced expression (9, 10)...

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Section: Resultsmentioning

confidence: 99%

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300-Fold Increase in Production of the Zn ²⁺ -Dependent Dechlorinase TrzN in Soluble Form via Apoenzyme Stabilization

Jackson

Coppin

Carr

et al. 2014

Appl Environ Microbiol

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“…Placement of the ametryn and atratone in the active site also agreed with initial docking studies with only slight differences in the conformation of the alkyl groups (data not shown). Note that a published homology modeled structure of AtzA showed the alkyl groups in the opposite orientation with respect to the conserved active-site glutamate (40). This conformation is not likely as one of the N-alkyl binding pockets is clearly smaller than the other.…”

Section: Table 3 Kinetic Constants For Wild-type and Mutant Trzn Withmentioning

confidence: 99%

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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“…Combining rational engineering principles with an evolutionary approach has proven particularly powerful: small focused libraries are synthesised, guided by structure-function information, and then screened/ selected for properties of interest 10 . Such strategies have been used to alter properties such as stereospecificity, expression level 11 and Michaelis constants (K M ) 12 and to overcome functional constraints, such as inhibition by substrates and/or products leading to improved reaction yields 13 .…”

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Hacking nature: genetic tools for reprograming enzymes

2017

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Enzymes have many modern industrial applications, from biomass decomposition in the production of biofuels to highly stereospecific biotransformations in pharmaceutical manufacture. The capacity to find or engineer enzymes with activities pertinent to specific applications has been essential for the growth of a multibillion dollar enzyme industry. Over the course of the past 50-60 years our capacity to address this issue has become increasingly sophisticated, supported by innumerable advances, from early discoveries such as the co-linearity of DNA and protein sequence 1 to modern computational technologies for enzyme design. The design of enzyme function is an exciting nexus of fundamental biochemical understanding and applied engineering. Herein, we will cover some of the methods used in discovery and design, including some 'next generation' tools.Traditionally, enzymes with useful biochemical properties have been sourced from nature, tapping into the natural diversity generated by evolution. Where known physiological functions are useful in an industrial setting, it is relatively simple to match an enzyme to an application (e.g. amylase-mediated glucose production from starch). Where novel functions are required, enrichment culturing of microbes can be used: for example, the recent isolation of bacteria capable of using nylon intermediates as a nitrogen source with potential utility in nylon manufacture (Figure 1) 3 . Non-culture based methods can also be applied to enzyme discovery, for example by exploiting the explosion of genetic information that followed the 'omics' revolution. Driven by technological advances in DNA sequencing, and computational power, this has provided an enormous resource, accessible by bioinformatic analysis, and leading to the discovery of enzymes with industrial applications, such as novel imine reductases for asymmetric organic synthesis 4 .Methods have also been developed to move beyond the repertoire of enzymes currently known in nature. One of the most prevalent approaches has been to use atomic information about structure and function to rationally redesign enzymes, often to expand or alter substrate range, change stereospecificity or alter physical however, the catalytic properties of such synthetic and semisynthetic enzymes can be improved by direction evolution and related methodologies 21,22 .In the approaches considered above, enzyme engineers have been content to explore the chemical space provided by the 20 canonical

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Catalytic Improvement and Evolution of Atrazine Chlorohydrolase

Cited by 58 publications

References 59 publications

300-Fold Increase in Production of the Zn ²⁺ -Dependent Dechlorinase TrzN in Soluble Form via Apoenzyme Stabilization

300-Fold Increase in Production of the Zn ²⁺ -Dependent Dechlorinase TrzN in Soluble Form via Apoenzyme Stabilization

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

Hacking nature: genetic tools for reprograming enzymes

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Catalytic Improvement and Evolution of Atrazine Chlorohydrolase

Cited by 58 publications

References 59 publications

300-Fold Increase in Production of the Zn 2+ -Dependent Dechlorinase TrzN in Soluble Form via Apoenzyme Stabilization

300-Fold Increase in Production of the Zn 2+ -Dependent Dechlorinase TrzN in Soluble Form via Apoenzyme Stabilization

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

Hacking nature: genetic tools for reprograming enzymes

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300-Fold Increase in Production of the Zn ²⁺ -Dependent Dechlorinase TrzN in Soluble Form via Apoenzyme Stabilization

300-Fold Increase in Production of the Zn ²⁺ -Dependent Dechlorinase TrzN in Soluble Form via Apoenzyme Stabilization