Functional Annotation of LigU as a 1,3-Allylic Isomerase during the Degradation of Lignin in the Protocatechuate 4,5-Cleavage Pathway from the Soil Bacterium <i>Sphingobium</i> sp. SYK‑6

<i>Sphingobium</i> sp. SYK-6 is a Gram-negative soil bacterium that contributes to the degradation of lignin. Lignin provides structural support and protection to plants as a complex aromatic heteropolymer. The lignin degradation pathway of guaiacyl moieties leads to the intermediate, protocatechuate (PCA), which is further degraded via the 4,5-cleavage pathway in which PCA is ultimately metabolized to pyruvate and oxaloacetate. In this pathway, LigI has been shown to catalyze the hydrolysis of 2-pyrone-4,6-dicarboxylate to (4<i>E</i>)-oxalomesaconate (OMA). Here we have demonstrated, using ¹H and ¹³C nuclear magnetic resonance spectroscopy, that LigU catalyzes the isomerization of the double bond between C4 and C5 in (4<i>E</i>)-OMA to (3<i>Z</i>)-2-keto-4-carboxy-3-hexenedioate (KCH), where the double bond has migrated to be between C3 and C4 via a 1,3-allylic isomerization. LigU is most closely related in amino acid sequence to methylaconitate isomerase (PrpF) from <i>Shewanella oneidensis</i> and methylitaconate-Δ-isomerase (Mii) from <i>Eubacterium barkeri.</i> The kinetic constants for the isomerization of OMA to KCH by LigU at pH 8.0 were determined to be 1300 ± 120 s^–1 and (7.7 ± 1.5) × 10⁶ M^–1 s^–1 for <i>k</i>_cat and <i>k</i>_cat/<i>K</i>_m, respectively. We have also shown that the product of the LigU-catalyzed reaction is the preferred substrate for the LigJ hydratase. In this reaction, LigJ catalyzes the hydration of KCH to 4-carboxy-4-hydroxy-2-oxoadipate

Discovery of a Previously Unrecognized Ribonuclease from <i>Escherichia coli</i> That Hydrolyzes 5′-Phosphorylated Fragments of RNA

TrpH or YciV (locus tag b1266) from <i>Escherichia coli</i> is annotated as a protein of unknown function that belongs to the polymerase and histidinol phosphatase (PHP) family of proteins in the UniProt and NCBI databases. Enzymes from the PHP family have been shown to hydrolyze organophosphoesters using divalent metal ion cofactors at the active site. We found that TrpH is capable of hydrolyzing the 3′-phosphate from 3′,5′-bis-phosphonucleotides. The enzyme will also sequentially hydrolyze 5′-phosphomononucleotides from 5′-phosphorylated RNA and DNA oligonucleotides, with no specificity toward the identity of the nucleotide base. The enzyme will not hydrolyze RNA or DNA oligonucleotides that are unphosphorylated at the 5′-end of the substrate, but it makes no difference whether the 3′-end of the oligonucleotide is phosphorylated. These results are consistent with the sequential hydrolysis of 5′-phosphorylated mononucleotides from oligonucleotides in the 5′ → 3′ direction. The catalytic efficiencies for hydrolysis of 3′,5′-pAp, p­(Ap)­A, p­(Ap)₄A, and p­(dAp)₄dA were determined to be 1.8 × 10⁵, 9.0 × 10⁴, 4.6 × 10⁴, and 2.9 × 10³ M^–1 s^–1, respectively. TrpH was found to be more efficient at hydrolyzing RNA oligonucleotides than DNA oligonucleotides. This enzyme can also hydrolyze annealed DNA duplexes, albeit at a catalytic efficiency approximately 10-fold lower than that of the corresponding single-stranded oligonucleotides. TrpH is the first enzyme from <i>E. coli</i> that has been found to possess 5′ → 3′ exoribonuclease activity. We propose to name this enzyme RNase AM

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>_cat = 1.1 s^–1, <i>K</i>_m = 1.05 mM, and <i>k</i>_cat/<i>K</i>_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 (<i>k</i>_cat = 21 s^–1, <i>K</i>_m = 18 μM, and <i>k</i>_cat/<i>K</i>_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 <i>k</i>_cat = 2.1 s^–1, <i>K</i>_m = 69 μM, and <i>k</i>_cat/<i>K</i>_m = 3.1 × 10⁴ M^–1 s^–1

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>_P-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>_P-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

Biosynthesis of Nucleoside Diphosphoramidates in <i>Campylobacter jejuni</i>

<i>Campylobacter jejuni</i> is a pathogenic Gram-negative bacterium and a leading cause of food-borne gastroenteritis. <i>C. jejuni</i> produces a capsular polysaccharide (CPS) that contains a unique <i>O</i>-methyl phosphoramidate modification (MeOPN). Recently, the first step in the biosynthetic pathway for the assembly of the MeOPN modification to the CPS was elucidated. It was shown that the enzyme Cj1418 catalyzes the phosphorylation of the amide nitrogen of l-glutamine to form l-glutamine phosphate. In this investigation, the metabolic fate of l-glutamine phosphate was determined. The enzyme Cj1416 catalyzes the displacement of pyrophosphate from MgCTP by l-glutamine phosphate to form CDP-l-glutamine. The enzyme Cj1417 subsequently catalyzes the hydrolysis of CDP-l-glutamine to generate cytidine diphosphoramidate and l-glutamate. The structures of the two novel intermediates, CDP-l-glutamine and cytidine diphosphoramidate, were confirmed by ³¹P nuclear magnetic resonance spectroscopy and mass spectrometry. It is proposed that the enzyme Cj1416 be named CTP:phosphoglutamine cytidylyltransferase and that the enzyme Cj1417 be named γ-glutamyl-CDP-amidate hydrolase

Discovery of a Cyclic Phosphodiesterase That Catalyzes the Sequential Hydrolysis of Both Ester Bonds to Phosphorus

The bacterial C–P lyase pathway is responsible for the metabolism of unactivated organophosphonates under conditions of phosphate starvation. The cleavage of the C–P bond within ribose-1-methylphosphonate-5-phosphate to form methane and 5-phospho-ribose-1,2-cyclic phosphate (PRcP) is catalyzed by the radical SAM enzyme PhnJ. In <i>Escherichia coli</i> the cyclic phosphate product is hydrolyzed to ribose-1,5-bisphosphate by PhnP. In this study, we describe the discovery and characterization of an enzyme that can hydrolyze a cyclic phosphodiester directly to a vicinal diol and inorganic phosphate. With PRcP, this enzyme hydrolyzes the phosphate ester at carbon-1 of the ribose moiety to form ribose-2,5-bisphosphate, and then this intermediate is hydrolyzed to ribose-5-phosphate and inorganic phosphate. Ribose-1,5-bisphosphate is neither an intermediate nor a substrate for this enzyme. Orthologues of this enzyme are found in the human pathogens <i>Clostridium difficile</i> and <i>Eggerthella lenta</i>. We propose that this enzyme be called cyclic phosphate dihydrolase (cPDH) and be designated as PhnPP

Enzymatic Neutralization of the Chemical Warfare Agent VX: Evolution of Phosphotriesterase for Phosphorothiolate Hydrolysis

The V-type nerve agents (VX and VR) are among the most toxic substances known. The high toxicity and environmental persistence of VX make the development of novel decontamination methods particularly important. The enzyme phosphotriesterase (PTE) is capable of hydrolyzing VX but with an enzymatic efficiency more than 5 orders of magnitude lower than with its best substrate, paraoxon. PTE has previously proven amenable to directed evolution for the improvement of catalytic activity against selected compounds through the manipulation of active-site residues. Here, a series of sequential two-site mutational libraries encompassing 12 active-site residues of PTE was created. The libraries were screened for catalytic activity against a new VX analogue, DEVX, which contains the same thiolate leaving group of VX coupled to a diethoxyphosphate core rather than the ethoxymethylphosphonate core of VX. The evolved catalytic activity with DEVX was enhanced 26-fold relative to wild-type PTE. Further improvements were facilitated by targeted error-prone PCR mutagenesis of loop-7, and additional PTE variants were identified with up to a 78-fold increase in the rate of DEVX hydrolysis. The best mutant hydrolyzed the racemic nerve agent VX with a value of <i>k</i>_cat/<i>K</i>_m = 7 × 10⁴ M^–1 s^–1, a 230-fold improvement relative to wild-type PTE. The highest turnover number achieved by the mutants created for this investigation was 137 s^–1, an enhancement of 152-fold relative to wild-type PTE. The stereoselectivity for the hydrolysis of the two enantiomers of VX was relatively low. These engineered mutants of PTE are the best catalysts ever reported for the hydrolysis of nerve agent VX

l‑Galactose Metabolism in <i>Bacteroides vulgatus</i> from the Human Gut Microbiota

A previously unknown metabolic pathway for the utilization of l-galactose was discovered in a prevalent gut bacterium, <i>Bacteroides vulgatus</i>. The new pathway consists of three previously uncharacterized enzymes that were found to be responsible for the conversion of l-galactose to d-tagaturonate. Bvu0219 (l-galactose dehydrogenase) was determined to oxidize l-galactose to l-galactono-1,5-lactone with <i>k</i>_cat and <i>k</i>_cat<i>/K</i>_m values of 21 s^–1 and 2.0 × 10⁵ M^–1 s^–1, respectively. The kinetic product of Bvu0219 is rapidly converted nonenzymatically to the thermodynamically more stable l-galactono-1,4-lactone. Bvu0220 (l-galactono-1,5-lactonase) hydrolyzes both the kinetic and thermodynamic products of Bvu0219 to l-galactonate. However, l-galactono-1,5-lactone is estimated to be hydrolyzed 300-fold faster than its thermodynamically more stable counterpart, l-galactono-1,4-lactone. In the final step of this pathway, Bvu0222 (l-galactonate dehydrogenase) oxidizes l-galactonate to d-tagaturonate with <i>k</i>_cat and <i>k</i>_cat<i>/K</i>_m values of 0.6 s^–1 and 1.7 × 10⁴ M^–1 s^–1, respectively. In the reverse direction, d-tagaturonate is reduced to l-galactonate with values of <i>k</i>_cat and <i>k</i>_cat/<i>K</i>_m of 90 s^–1 and 1.6 × 10⁵ M^–1 s^–1, respectively. d-Tagaturonate is subsequently converted to d-glyceraldehyde and pyruvate through enzymes encoded within the degradation pathway for d-glucuronate and d-galacturonate

Subunit Interactions within the Carbon–Phosphorus Lyase Complex from Escherichia coli

Phosphonates are a large class of organophosphorus compounds with a characteristic carbon–phosphorus bond. The genes responsible for phosphonate utilization in Gram-negative bacteria are arranged in an operon of 14 genes. The carbon–phosphorus lyase complex, encoded by the genes <i>phnGHIJKLM</i>, catalyzes the cleavage of the stable carbon–phosphorus bond of organophosphonates to the corresponding hydrocarbon and inorganic phosphate. Recently, complexes of this enzyme containing five subunits (PhnG-H-I-J-K), four subunits (PhnG-H-I-J), and two subunits (PhnG-I) were purified after expression in Escherichia coli (Proc. Natl. Acad. Sci., U. S. A. 2011, 108, 11393). Here we demonstrated using mass spectrometry, ultracentrifugation, and chemical cross-linking experiments that these complexes are formed from a PhnG₂I₂ core that is further elaborated by the addition of two copies each of PhnH and PhnJ to generate PhnG₂H₂I₂J₂. This complex adds an additional subunit of PhnK to form PhnG₂H₂I₂J₂K. Chemical cross-linking of the five-component complex demonstrated that PhnJ physically interacts with both PhnG and PhnI. We were unable to demonstrate the interaction of PhnH or PhnK with any other subunits by chemical cross-linking. Hydrogen–deuterium exchange was utilized to probe for alterations in the dynamic properties of individual subunits within the various complexes. Significant regions of PhnG become less accessible to hydrogen/deuterium exchange from solvent within the PhnG₂I₂ complex compared with PhnG alone. Specific regions of PhnI exhibited significant differences in the H/D exchange rates in PhnG₂I₂ and PhnG₂H₂I₂J₂K