15 research outputs found
Transition State Analysis of the Reaction Catalyzed by the Phosphotriesterase from Sphingiobium sp. TCM1
Organophosphorus flame retardants are stable toxic compounds used in nearly all durable plastic products and are considered major emerging pollutants. The phosphotriesterase from Sphingobium sp. TCM1 (Sb-PTE) is one of the few enzymes known to be able to hydrolyze organophosphorus flame retardants such as triphenyl phosphate and tris(2-chloroethyl) phosphate. The effectiveness of Sb-PTE for the hydrolysis of these organophosphates appears to arise from its ability to hydrolyze unactivated alkyl and phenolic esters from the central phosphorus core. How Sb-PTE is able to catalyze the hydrolysis of the unactivated substituents is not known. To interrogate the catalytic hydrolysis mechanism of Sb-PTE, the pH dependence of the reaction and the effects of changing the solvent viscosity were determined. These experiments were complemented by measurement of the primary and secondary 18-oxygen isotope effects on substrate hydrolysis and a determination of the effects of changing the pKa of the leaving group on the magnitude of the rate constants for hydrolysis. Collectively, the results indicated that a single group must be ionized for nucleophilic attack and that a separate general acid is not involved in protonation of the leaving group. The Brønsted analysis and the heavy atom kinetic isotope effects are consistent with an early associative transition state with subsequent proton transfers not being rate limiting. A novel binding mode of the substrate to the binuclear metal center and a catalytic mechanism are proposed to explain the unusual ability of Sb-PTE to hydrolyze unactivated esters from a wide range of organophosphate substrates
Functional characterization of two PLP-dependent enzymes involved in capsular polysaccharide biosynthesis from campylobacter jejuni
Campylobacter jejuni is a Gram-negative, pathogenic bacterium that causes campylobacteriosis, a form of gastroenteritis. C. jejuni is the most frequent cause of food-borne illness in the world, surpassing Salmonella and E. coli. Coating the surface of C. jejuni is a layer of sugar molecules known as the capsular polysaccharide that, in C. jejuni NCTC 11168, is composed of a repeating unit of d-glycero-l-gluco-heptose, d-glucuronic acid, d-N-acetyl-galactosamine, and d-ribose. The d-glucuronic acid moiety is further amidated with either serinol or ethanolamine. It is unknown how these modifications are synthesized and attached to the polysaccharide. Here, we report the catalytic activities of two previously uncharacterized, pyridoxal phosphate (PLP)-dependent enzymes, Cj1436 and Cj1437, from C. jejuni NCTC 11168. Using a combination of mass spectrometry and nuclear magnetic resonance, we determined that Cj1436 catalyzes the decarboxylation of l-serine phosphate to ethanolamine phosphate. Cj1437 was shown to catalyze the transamination of dihydroxyacetone phosphate to (S)-serinol phosphate in the presence of l-glutamate. The probable routes to the ultimate formation of the glucuronamide substructures in the capsular polysaccharides of C. jejuni are discussed
Biosynthesis of UDP-β‑l‑Arabinofuranoside for the Capsular Polysaccharides of Campylobacter jejuni
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
Discovery of a Kojibiose Phosphorylase in <i>Escherichia coli</i> K‑12
The substrate profiles
for three uncharacterized enzymes (YcjM,
YcjT, and YcjU) that are expressed from a cluster of 12 genes (<i>ycjM-W</i> and <i>ompG</i>) of unknown function in <i>Escherichia coli</i> 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 (<i>k</i><sub>cat</sub> = 1.1 s<sup>–1</sup>, <i>K</i><sub>m</sub> = 1.05 mM, and <i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub> = 1.12 × 10<sup>3</sup> M<sup>–1</sup> s<sup>–1</sup>). 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
(<i>k</i><sub>cat</sub> = 21 s<sup>–1</sup>, <i>K</i><sub>m</sub> = 18 μM, and <i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub> = 1.1 × 10<sup>6</sup> M<sup>–1</sup> s<sup>–1</sup>) 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 <i>k</i><sub>cat</sub> = 2.1 s<sup>–1</sup>, <i>K</i><sub>m</sub> = 69 μM, and <i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub> = 3.1 × 10<sup>4</sup> M<sup>–1</sup> s<sup>–1</sup>
Multiple Reaction Products from the Hydrolysis of Chiral and Prochiral Organophosphate Substrates by the Phosphotriesterase from <i>Sphingobium</i> sp. TCM1
The
phosphotriesterase from <i>Sphingobium</i> sp. TCM1
(<i>Sb</i>-PTE) is notable for its ability to hydrolyze
organophosphates that are not substrates for other enzymes. In an
attempt to determine the catalytic properties of <i>Sb</i>-PTE for hydrolysis of chiral phosphotriesters, we discovered that
multiple phosphodiester products are formed from a single substrate.
For example, <i>Sb</i>-PTE catalyzes the hydrolysis of the <i>R</i><sub>P</sub>-enantiomer of methyl cyclohexyl <i>p</i>-nitrophenyl phosphate with exclusive formation of methyl cyclohexyl
phosphate. However, the enzyme catalyzes hydrolysis of the <i>S</i><sub>P</sub>-enantiomer of this substrate to an equal mixture
of methyl cyclohexyl phosphate and cyclohexyl <i>p</i>-nitrophenyl
phosphate products. The ability of this enzyme to catalyze the hydrolysis
of a methyl ester at the same rate as the hydrolysis of a <i>p</i>-nitrophenyl ester contained within the same substrate
is remarkable. The overall scope of the stereoselective properties
of this enzyme is addressed with a library of chiral and prochiral
substrates
Functional Annotation and Three-Dimensional Structure of an Incorrectly Annotated Dihydroorotase from cog3964 in the Amidohydrolase Superfamily
The substrate specificities of two incorrectly annotated enzymes belonging to cog3964 from the amidohydrolase superfamily were determined. This group of enzymes are currently misannotated as either dihydroorotases or adenine deaminases. Atu3266 from Agrobacterium tumefaciens C58 and Oant2987 from Ochrobactrum anthropi ATCC 49188 were found to catalyze the hydrolysis of acetyl-(R)-mandelate and similar esters with values of k(cat)/K(m) that exceed 10(5) M(-1) s(-1). These enzymes do not catalyze the deamination of adenine or the hydrolysis of dihydroorotate. Atu3266 was crystallized and the structure determined to a resolution of 2.62 Å. The protein folds as a distorted (β/α)(8) barrel and binds two zincs in the active site. The substrate profile was determined via a combination of computational docking to the three-dimensional structure of Atu3266 and screening of a highly focused library of potential substrates. The initial weak hit was the hydrolysis of N-acetyl-D-serine (k(cat)/K(m) = 4 M(-1) s(-1)). This was followed by the progressive identification of acetyl-(R)-glycerate (k(cat)/K(m) = 4 × 10(2) M(-1) s(-1)), acetyl glycolate (k(cat)/K(m) = 1.3 × 10(4) M(-1) s(-1)), and ultimately acetyl-(R)-mandelate (k(cat)/K(m) = 2.8 × 10(5) M(-1) s(-1))
Functional Annotation and Three-Dimensional Structure of an Incorrectly Annotated Dihydroorotase from cog3964 in the Amidohydrolase Superfamily
The substrate specificities of two incorrectly annotated
enzymes
belonging to cog3964 from the amidohydrolase superfamily were determined.
This group of enzymes are currently misannotated as either dihydroorotases
or adenine deaminases. Atu3266 from <i>Agrobacterium tumefaciens</i> C58 and Oant2987 from <i>Ochrobactrum anthropi</i> ATCC
49188 were found to catalyze the hydrolysis of acetyl-(<i>R</i>)-mandelate and similar esters with values of <i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub> that exceed 10<sup>5</sup> M<sup>–1</sup> s<sup>–1</sup>. These enzymes do not
catalyze the deamination of adenine or the hydrolysis of dihydroorotate.
Atu3266 was crystallized and the structure determined to a resolution
of 2.62 Å. The protein folds as a distorted (β/α)<sub>8</sub> barrel and binds two zincs in the active site. The substrate
profile was determined via a combination of computational docking
to the three-dimensional structure of Atu3266 and screening of a highly
focused library of potential substrates. The initial weak hit was
the hydrolysis of <i>N</i>-acetyl-d-serine (<i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub> = 4 M<sup>–1</sup> s<sup>–1</sup>). This was followed by the
progressive identification of acetyl-(<i>R</i>)-glycerate
(<i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub> =
4 × 10<sup>2</sup> M<sup>–1</sup> s<sup>–1</sup>), acetyl glycolate (<i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub> = 1.3 × 10<sup>4</sup> M<sup>–1</sup> s<sup>–1</sup>), and ultimately acetyl-(<i>R</i>)-mandelate
(<i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub> =
2.8 × 10<sup>5</sup> M<sup>–1</sup> s<sup>–1</sup>)