22 research outputs found
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 <sup>1</sup>H and <sup>13</sup>C 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<sup>–1</sup> and (7.7 ± 1.5) × 10<sup>6</sup> M<sup>–1</sup> s<sup>–1</sup> for <i>k</i><sub>cat</sub> and <i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub>, 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,
pÂ(Ap)ÂA, pÂ(Ap)<sub>4</sub>A, and pÂ(dAp)<sub>4</sub>dA were determined
to be 1.8 × 10<sup>5</sup>, 9.0 × 10<sup>4</sup>, 4.6 ×
10<sup>4</sup>, and 2.9 × 10<sup>3</sup> M<sup>–1</sup> s<sup>–1</sup>, 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><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
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 <sup>31</sup>P 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><sub>cat</sub>/<i>K</i><sub>m</sub> = 7 × 10<sup>4</sup> M<sup>–1</sup> s<sup>–1</sup>, a 230-fold improvement relative to wild-type PTE.
The highest turnover number achieved by the mutants created for this
investigation was 137 s<sup>–1</sup>, 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><sub>cat</sub> and <i>k</i><sub>cat</sub><i>/K</i><sub>m</sub> values of 21 s<sup>–1</sup> and 2.0
× 10<sup>5</sup> M<sup>–1</sup> s<sup>–1</sup>,
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><sub>cat</sub> and <i>k</i><sub>cat</sub><i>/K</i><sub>m</sub> values of
0.6 s<sup>–1</sup> and 1.7 × 10<sup>4</sup> M<sup>–1</sup> s<sup>–1</sup>, respectively. In the reverse direction, d-tagaturonate is reduced to l-galactonate with values
of <i>k</i><sub>cat</sub> and <i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub> of 90 s<sup>–1</sup> and 1.6
× 10<sup>5</sup> M<sup>–1</sup> s<sup>–1</sup>,
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 PhnG<sub>2</sub>I<sub>2</sub> core that is further elaborated by the addition
of two copies each of PhnH and PhnJ to generate PhnG<sub>2</sub>H<sub>2</sub>I<sub>2</sub>J<sub>2</sub>. This complex adds an additional
subunit of PhnK to form PhnG<sub>2</sub>H<sub>2</sub>I<sub>2</sub>J<sub>2</sub>K. 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 PhnG<sub>2</sub>I<sub>2</sub> complex compared
with PhnG alone. Specific regions of PhnI exhibited significant differences
in the H/D exchange rates in PhnG<sub>2</sub>I<sub>2</sub> and PhnG<sub>2</sub>H<sub>2</sub>I<sub>2</sub>J<sub>2</sub>K