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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
2.
Catalytic mechanisms for phosphotriesterases Bigley, Andrew N.; Raushel, Frank M.
Biochimica Et Biophysica Acta - Proteins And Proteomics,
01/2013, Letnik:
1834, Številka:
1
Journal Article
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Phosphotriesters are one class of highly toxic synthetic compounds known as organophosphates. Wide spread usage of organophosphates as insecticides as well as nerve agents has lead to numerous ...efforts to identify enzymes capable of detoxifying them. A wide array of enzymes has been found to have phosphotriesterase activity including phosphotriesterase (PTE), methyl parathion hydrolase (MPH), organophosphorus acid anhydrolase (OPAA), diisopropylfluorophosphatase (DFP), and paraoxonase 1 (PON1). These enzymes differ widely in protein sequence and three-dimensional structure, as well as in catalytic mechanism, but they also share several common features. All of the enzymes identified as phosphotriesterases are metal-dependent hydrolases that contain a hydrophobic active site with three discrete binding pockets to accommodate the substrate ester groups. Activation of the substrate phosphorus center is achieved by a direct interaction between the phosphoryl oxygen and a divalent metal in the active site. The mechanistic details of the hydrolytic reaction differ among the various enzymes with both direct attack of a hydroxide as well as covalent catalysis being found. This article is part of a Special Issue entitled: Chemistry and mechanism of phosphatases, diesterases and triesterases.
► The role of binuclear metal clusters in phosphotriester hydrolysis. ► The mechanism of action for the hydrolysis of oganophosphate triesters. ► Variations in protein structure for various phosphotriesterases.
The organophosphorus chemical warfare agents were initially synthesized in the 1930's and are some of the most toxic compounds ever discovered. The standard means of decontamination are either harsh ...chemical hydrolysis or high temperature incineration. Given the continued use of chemical warfare agents there are ongoing efforts to develop gentle environmentally friendly means of decontamination and medical counter measures to chemical warfare agent intoxication. Enzymatic decontamination offers the benefits of extreme specificity and mild conditions, allowing their use for both environmental and medical applications. The most promising enzyme for decontamination of the organophosphorus chemical warfare agents is the enzyme phosphotriesterase from Pseudomonas diminuta. However, the catalytic activity of the wild-type enzyme with the chemical warfare agents falls far below that seen with its best substrates, and its stereochemical preference is for the less toxic enantiomer of the chiral phosphorus center found in most chemical warfare agents. Rational design efforts have succeeded in the dramatic improvement of the stereochemical preference of PTE for the more toxic enantiomers. Directed evolution experiments, including site-saturation mutagenesis, targeted error-prone PCR, computational design, and quantitative library analysis, have systematically improved the catalytic activity against the chemical warfare nerve agents. These efforts have resulted in greater than 4-orders of magnitude improvement in catalytic activity and have led to the identification of variants that are highly effective at detoxifying both G-type and V-type nerve agents. The best of these variants have the ability to prevent intoxication when delivered as a post-exposure treatment for VX and as a pre-exposure treatment for G-agent intoxication with observed protective factors up to 60-fold. Combining the best variant, H257Y/L303T, with a PCB polymer coating has enabled the development of a long lasting circulating prophylactic treatment that is highly effective against sarin.
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•PTE has been evolved for chemical warfare agent decontamination.•High activity and broad specificity against G-agents has been attained.•High activity and stereospecificity against VX and VR has been accomplished.•In vivo protection against both G-agents and VX has been demonstrated.•PTE has been adapted as a long circulations prophylactic against G-agents.
The amidohydrolase superfamily comprises a remarkable set of enzymes that catalyze the hydrolysis of a wide range of substrates bearing amide or ester functional groups at carbon and phosphorus ...centers. The most salient structural landmark for this family of hydrolytic enzymes is a mononuclear or binuclear metal center embedded within the confines of a (β/α)8-barrel structural fold. Seven variations in the identity of the specific amino acids that function as the direct metal ligands have been structurally characterized by X-ray crystallography. The metal center in this enzyme superfamily has a dual functionality in the expression of the overall catalytic activity. The scissile bond of the substrate must be activated for bond cleavage, and the hydrolytic water molecule must be deprotonated for nucleophilic attack. In all cases, the nucleophilic water molecule is activated through complexation with a mononuclear or binuclear metal center. In the binuclear metal centers, the carbonyl and phosphoryl groups of the substrates are polarized through Lewis acid catalysis via complexation with the β-metal ion, while the hydrolytic water molecule is activated for nucleophilic attack by interaction with the α-metal ion. In the mononuclear metal centers, the substrate is activated by proton transfer from the active site, and the water is activated by metal ligation and general base catalysis. The substrate diversity is dictated by the conformational restrictions imposed by the eight loops that extend from the ends of the eight β-strands.
The substrate profiles for three uncharacterized enzymes (YcjM, YcjT, and YcjU) that are expressed from a cluster of 12 genes (ycjM-W and ompG) of unknown function in Escherichia coli K-12 were ...determined. Through a comprehensive bioinformatic and steady-state kinetic analysis, the catalytic function of YcjT was determined to be kojibiose phosphorylase. In the presence of saturating phosphate and kojibiose (α-(1,2)-d-glucose-d-glucose), this enzyme catalyzes the formation of d-glucose and β-d-glucose-1-phosphate (k cat = 1.1 s–1, K m = 1.05 mM, and k cat/K m = 1.12 × 103 M–1 s–1). Additionally, it was also shown that in the presence of β-d-glucose-1-phosphate, YcjT can catalyze the formation of other disaccharides using 1,5-anhydro-d-glucitol, l-sorbose, d-sorbitol, or l-iditol as a substitute for d-glucose. Kojibiose is a component of cell wall lipoteichoic acids in Gram-positive bacteria and is of interest as a potential low-calorie sweetener and prebiotic. YcjU was determined to be a β-phosphoglucomutase that catalyzes the isomerization of β-d-glucose-1-phosphate (k cat = 21 s–1, K m = 18 μM, and k cat/K m = 1.1 × 106 M–1 s–1) to d-glucose-6-phosphate. YcjU was also shown to exhibit catalytic activity with β-d-allose-1-phosphate, β-d-mannose-1-phosphate, and β-d-galactose-1-phosphate. YcjM catalyzes the phosphorolysis of α-(1,2)-d-glucose-d-glycerate with a k cat = 2.1 s–1, K m = 69 μM, and k cat/K m = 3.1 × 104 M–1 s–1.
ProTides are nucleotide analogues used for the treatment of specific viral infections. These compounds consist of a masked nucleotide that undergoes in vivo enzymatic and spontaneous chemical ...transformations to generate a free mononucleotide that is ultimately transformed to the pharmaceutically active triphosphorylated drug. The three FDA approved ProTides are composed of a phosphoramidate (P–N) core coupled with a nucleoside analogue, phenol, and an l-alanyl carboxylate ester. The previously proposed mechanism of activation postulates the existence of an unstable 5-membered mixed anhydride cyclic intermediate formed from the direct attack of the carboxylate group of the l-alanyl moiety with expulsion of phenol. The mixed anhydride cyclic intermediate is further postulated to undergo spontaneous hydrolysis to form a linear l-alanyl phosphoramidate product. In the proposed mechanism of activation, the 5-membered mixed anhydride intermediate has been detected previously using mass spectrometry, but the specific site of nucleophilic attack by water (P–O versus C–O) has not been determined. To further interrogate the mechanism for hydrolysis of the putative 5-membered cyclic intermediate formed during ProTide activation, the reaction was conducted in 18O-labeled water using a ProTide analogue that could be activated by carboxypeptidase Y. Mass spectrometry and 31P NMR spectroscopy were used to demonstrate that the hydrolysis of the mixed anhydride 5-membered intermediate occurs with exclusive attack at the phosphorus center.
Campylobacter jejuni is a human pathogen and a leading cause of food poisoning in North America and Europe. The exterior surface of the bacterial cell wall is attached to a polymeric coat of sugar ...molecules known as the capsular polysaccharide (CPS) that helps protect the organism from the host immune response. The CPS is composed of a repeating sequence of common and unusual sugar residues. In the HS:11 serotype of C. jejuni, we identified two enzymes in the gene cluster for CPS formation that are utilized for the biosynthesis of UDP-α-N-acetyl-d-mannosaminuronic acid (UDP-ManNAcA). In the first step, UDP-α-N-acetyl-d-glucosamine (UDP-GlcNAc) is epimerized at C2 to form UDP-α-N-acetyl-d-mannosamine (UDP-ManNAc). This product is then oxidized by a NAD+-dependent C6-dehydrogenase to form UDP-ManNAcA. In the HS:6 serotype (C. jejuni strain 81116), we identified three enzymes that are required for the biosynthesis of CMP-β-N-acetyl-d-neuraminic acid (CMP-Neu5Ac). In the first step, UDP-GlcNAc is epimerized at C2 and subsequently hydrolyzed to form N-acetyl-d-mannosamine (ManNAc) with the release of UDP. This product is then condensed with PEP by N-acetyl-d-neuraminate synthase to form N-acetyl-d-neuraminic acid (Neu5Ac). In the final step, CMP-N-acetyl-d-neuraminic acid synthase utilizes CTP to convert this product into CMP-Neu5Ac. A bioinformatic analysis of these five enzymes from C. jejuni serotypes HS:11 and HS:6 identified other bacterial species that can produce UDP-ManNAcA or CMP-Neu5Ac for CPS formation.
Campylobacter jejuni is the leading cause of food poisoning in the United States and Europe. A capsular polysaccharide that coats the exterior of the bacterium helps evade the host immune system. At ...least 33 different strains of C. jejuni have been identified, and the chemical structures of 12 different capsular polysaccharides (CPSs) have been characterized from various serotypes. Thus far, 10 different heptose sugars have been found in the chemically characterized CPSs, and each of these are currently thought to originate from the modification of GDP-d-glycero-d-manno-heptose by the successive action of 4,6-dehydratase (or C4-dehydrogenase), C3- or C3/C5-epimerase, and C4-reductase. Within the sequenced strains of C. jejuni, we have identified 25 different C4-reductases that cluster into nine groups at a sequence identity of >90%. Eight of the proteins from seven different clusters were purified, and their product profiles were determined with GDP-6-deoxy-4-keto-heptose substrates using NMR and ESI mass spectrometry. The isolated products included GDP-6-deoxy-l-gluco-heptose (serotype HS:2), GDP-6-deoxy-l-galacto-heptose (serotype HS:42), GDP-6-deoxy-l-gulo-heptose (serotype HS:15), GDP-6-deoxy-d-ido-heptose (serotypes HS:3, HS:4, and HS:33), GDP-6-deoxy-d-manno-heptose (serotype HS:53), and GDP-6-deoxy-d-altro-heptose (serotype HS:23/36). Based on these observations, the product specificity can be reliably predicted for 14 additional C4-reductases from C. jejuni. The remaining three C4-reductases are highly likely to be required for the biosynthesis of 3,6-dideoxy-heptose products.
Campylobacter jejuni is the leading cause of food poisoning in North America and Europe. The exterior surface of this bacterium is coated with a capsular polysaccharide (CPS) which enables adherence ...to the host epithelial cells and evasion of the host immune system. Many strains of C. jejuni can be differentiated from one another by changes in the sequence of the carbohydrates found within the CPS. The CPS structures of serotypes HS:15 and HS:41 of C. jejuni were chemically characterized and found to contain an l-arabinofuranoside moiety in the repeating CPS sequence. Sequence similarity and genome neighborhood networks were used to identify the putative gene cluster within the HS:15 serotype for the biosynthesis of the l-arabinofuranoside fragment. The first enzyme (HS:15.18) in the pathway was found to catalyze the NAD+-dependent oxidation of UDP-α-d-glucose to UDP-α-d-glucuronate, while the second enzyme (HS:15.19) catalyzes the NAD+-dependent decarboxylation of this product to form UDP-α-d-xylose. The UDP-α-d-xylose is then epimerized at C4 by the third enzyme (HS:15.17) to produce UDP-β-l-arabinopyranoside. In the last step, HS:15.16 catalyzes the FADH2-dependent conversion of UDP-β-l-arabinopyranoside into UDP-β-l-arabinofuranoside. The UDP-β-l-arabinopyranoside mutase catalyzed reaction was further interrogated by measurement of a positional isotope exchange reaction within 18O-UDP-β-l-arabinopyranoside.
Campylobacter jejuni is the leading cause of food poisoning in the United States. Surrounding the exterior surface of this bacterium is a capsular polysaccharide (CPS) that helps protect the organism ...from the host immune system. The CPS is composed of a repeating sequence of common and unusual sugar residues, including relatively rare heptoses. In the HS:5 serotype, we identified four enzymes required for the biosynthesis of GDP-3,6-dideoxy-β-l-ribo-heptose. In the first step, GDP-d-glycero-α-d-manno-heptose is dehydrated to form GDP-6-deoxy-4-keto-α-d-lyxo-heptose. This product is then dehydrated by a pyridoxal phosphate-dependent C3-dehydratase to form GDP-3,6-dideoxy-4-keto-α-d-threo-heptose before being epimerized at C5 to generate GDP-3,6-dideoxy-4-keto-β-l-erythro-heptose. In the final step, a C4-reductase uses NADPH to convert this product to GDP-3,6-dideoxy-β-l-ribo-heptose. These results are at variance with the previous report of 3,6-dideoxy-d-ribo-heptose in the CPS from serotype HS:5 of C. jejuni. We also demonstrated that GDP-3,6-dideoxy-β-l-xylo-heptose is formed using the corresponding enzymes found in the gene cluster from serotype HS:11 of C. jejuni. The utilization of different C4-reductases from other serotypes of C. jejuni enabled the formation of GDP-3,6-dideoxy-α-d-arabino-heptose and GDP-3,6-dideoxy-α-d-lyxo-heptose.
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