The structure of an enzyme–product complex reveals the critical role of a terminal hydroxide nucleophile in the bacterial phosphotriesterase mechanism

Jackson, Colin J.; Kim, Hye-Kyung; Carr, P.D.; Liu, Jianwei; Ollis, David L.

doi:10.1016/j.bbapap.2005.06.008

Cited by 84 publications

(109 citation statements)

References 26 publications

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“…Figure 5 shows pH-rate profiles for the catalyzed hydrolysis of paraoxon by wild-type arPTE and the unnatural variants over a pH range of 4.0 -10.0 (Table 1). All variants exhibit acidic dissociation constants with pK e and pK es values between 4.0 and 5.0, consistent with previous results for native wild-type arPTE, 21 which indicates a titration of the rate-limiting step from catalysis (k 3 ) to a physical step (product release, k 5 ), as described previously. 19 However, unlike wild-type and Tyr309Mco arPTE, the Tyr309Hco variant undergoes a second deprotonation event at basic pH.…”

Section: Mechanistic Analysis Of Unnatural Phosphotriesterasessupporting

confidence: 91%

Improving a Natural Enzyme Activity through Incorporation of Unnatural Amino Acids

Ugwumba

Ozawa

et al. 2010

J. Am. Chem. Soc.

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Abstract:The bacterial phosphotriesterases catalyze hydrolysis of the pesticide paraoxon with very fast turnover rates and are thought to be near to their evolutionary limit for this activity. To test whether the naturally evolved turnover rate could be improved through the incorporation of unnatural amino acids and to probe the role of peripheral active site residues in nonchemical steps of the catalytic cycle (substrate binding and product release), we replaced the naturally occurring tyrosine amino acid at position 309 with unnatural L- (7-hydroxycoumarin-4-yl)ethylglycine (Hco) and L-(7-methylcoumarin-4-yl)ethylglycine amino acids, as well as leucine, phenylalanine, and tryptophan. Kinetic analysis suggests that the 7-hydroxyl group of Hco, particularly in its deprotonated state, contributes to an increase in the rate-limiting product release step of substrate turnover as a result of its electrostatic repulsion of the negatively charged 4-nitrophenolate product of paraoxon hydrolysis. The 8-11-fold improvement of this already highly efficient catalyst through a single rationally designed mutation using an unnatural amino acid stands in contrast to the difficulty in improving this native activity through screening hundreds of thousands of mutants with natural amino acids. These results demonstrate that designer amino acids provide easy access to new and valuable sequence and functional space for the engineering and evolution of existing enzyme functions.

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Section: Mechanistic Analysis Of Unnatural Phosphotriesterasessupporting

confidence: 91%

Improving a Natural Enzyme Activity through Incorporation of Unnatural Amino Acids

Ugwumba

Ozawa

et al. 2010

J. Am. Chem. Soc.

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“…The catalytic mechanism is thought to proceed via direct in-line nucleophilic attack from a water molecule, activated through its interaction with the Fe 2+ ion, at the electrophilic phosphorus of the substrate, which coordinates to the Zn 2+ ion [61]. Considering that organophosphate pesticides are synthetic compounds that have only existed in the environment since the 1950s, the rapid evolution of near diffusion-limited turnover rates in these enzymes is noteworthy.…”

Section: +mentioning

confidence: 99%

The enzymatic basis for pesticide bioremediation

et al. 2008

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“…Several OPdegrading enzymes have already been studied; however, the OP hydrolases most studied are the degrading enzyme from Pseudomonas diminuta (OPH) and the one from Agrobacterium radiobacter (OpdA) (Horne et al, 2006). These enzymes take part of a large family that require Figure 6: Crystalline structure of OpdA from Agrobacterium radiobacter with bound product dimethylthiophosphate (PDB ID: 2D2G), and representation of its active site (Jackson et al, 2005). two metal ions like Zn 2+ (OPH) or Co 2+ (OpdA) in α and β sites for the hydrolytic reaction process; both enzymes have a binuclear metal center and are promising in studies of OPS degradation (Jacquet et al, 2016;Schenk et al, 2012).…”

Section: Enzymatic Biodegradation Phosphotriesterase From Agrobacterimentioning

confidence: 99%

“…According to structural studies, the two metal ions coordinated in both (OpdA and OPH) enzymes interact with a carboxylated lysine (Lys169) residue and a hydroxide ion or water molecule (see Figure 6) (Ely et al, 2010;Jackson et al, 2005).…”

Section: Enzymatic Biodegradation Phosphotriesterase From Agrobacterimentioning

confidence: 99%

Enzimas degradantes de organofosforados: Base molecular e perspectivas para biorremediação enzimática de agroquímicos

et al. 2017

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RESUMO Muitos compostos organofosforados (OP) são utilizados até hoje na agricultura como pesticidas e, infelizmente, como agentes de guerra química (ou agentes dos nervos) também. Os pesticidas organofosforados e os agentes dos nervos são moléculas extremamente tóxicas, uma vez que atuam como inibidores da enzima Acetilcolinesterase (AChE). O efeito mais preocupante da exposição a estes compostos é a toxicidade colinérgica aguda, ou seja, a perda de coordenação muscular. Uma vez que o indivíduo se contamina, o processo de intoxicação começa através da ligação do OP no sítio ativo da enzima AChE inativando-a. Os tratamentos atuais para pessoas expostas a baixas doses de OP podem ser realizados com atropina, oximas e benzodiazepínicos. Processos de remediação importantes envolvem o emprego de técnicas de biorremediação utilizando diferentes enzimas degradantes, como a Fosfotriesterase da Agrobacterium radiobacter e SMP-30. Devido ao elevado número de intoxicações anualmente, é crucial buscar métodos de tratamento mais potentes e eficazes, e nesta linha, as técnicas envolvendo biorremediação parecem ser bastante promissoras para este propósito.

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The structure of an enzyme–product complex reveals the critical role of a terminal hydroxide nucleophile in the bacterial phosphotriesterase mechanism

Cited by 84 publications

References 26 publications

Improving a Natural Enzyme Activity through Incorporation of Unnatural Amino Acids

Improving a Natural Enzyme Activity through Incorporation of Unnatural Amino Acids

The enzymatic basis for pesticide bioremediation

Enzimas degradantes de organofosforados: Base molecular e perspectivas para biorremediação enzimática de agroquímicos

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