4 research outputs found
Discovery of a Novel l‑Lyxonate Degradation Pathway in <i>Pseudomonas aeruginosa</i> PAO1
The l-lyxonate dehydratase (LyxD) <i>in vitro</i> enzymatic
activity and <i>in vivo</i> metabolic function were assigned
to members of an isofunctional family within the mandelate racemase
(MR) subgroup of the enolase superfamily. This study combined <i>in vitro</i> and <i>in vivo</i> data to confirm that
the dehydration of l-lyxonate is the biological role of the
members of this family. <i>In vitro</i> kinetic experiments
revealed catalytic efficiencies of ∼10<sup>4</sup> M<sup>–1</sup> s<sup>–1</sup> as previously observed for members of other
families in the MR subgroup. Growth studies revealed that l-lyxonate is a carbon source for <i>Pseudomonas aeruginosa</i> PAO1; transcriptomics using qRT-PCR established that the gene encoding
LyxD as well as several other conserved proximal genes were upregulated
in cells grown on l-lyxonate. The proximal genes were shown
to be involved in a pathway for the degradation of l-lyxonate,
in which the first step is dehydration by LyxD followed by dehydration
of the 2-keto-3-deoxy-l-lyxonate product by 2-keto-3-deoxy-l-lyxonate dehydratase to yield α-ketoglutarate semialdehyde.
In the final step, α-ketoglutarate semialdehyde is oxidized
by a dehydrogenase to α-ketoglutarate, an intermediate in the
citric acid cycle. An X-ray structure for the LyxD from <i>Labrenzia
aggregata</i> IAM 12614 with Mg<sup>2+</sup> in the active site
was determined that confirmed the expectation based on sequence alignments
that LyxDs possess a conserved catalytic His-Asp dyad at the end of
seventh and sixth β-strands of the (β/α)<sub>7</sub>β-barrel domain as well as a conserved KxR motif at the end
of second β-strand; substitutions for His 316 or Arg 179 inactivated
the enzyme. This is the first example of both the LyxD function in
the enolase superfamily and a pathway for the catabolism of l-lyxonate
Discovery of Function in the Enolase Superfamily: d‑Mannonate and d‑Gluconate Dehydratases in the d‑Mannonate Dehydratase Subgroup
The
continued increase in the size of the protein sequence databases
as a result of advances in genome sequencing technology is overwhelming
the ability to perform experimental characterization of function.
Consequently, functions are assigned to the vast majority of proteins
via automated, homology-based methods, with the result that as many
as 50% are incorrectly annotated or unannotated (Schnoes et al. PLoS Comput. Biol. 2009, 5 (12), e1000605). This manuscript describes a
study of the d-mannonate dehydratase (ManD) subgroup of the
enolase superfamily (ENS) to investigate how function diverges as
sequence diverges. Previously, one member of the subgroup had been
experimentally characterized as ManD [dehydration of d-mannonate
to 2-keto-3-deoxy-d-mannonate (equivalently, 2-keto-3-deoxy-d-gluconate)]. In this study, 42 additional members were characterized
to sample sequence–function space in the ManD subgroup. These
were found to differ in both catalytic efficiency and substrate specificity:
(1) high efficiency (<i>k</i><sub>cat</sub>/<i>K</i><sub>M</sub> = 10<sup>3</sup> to 10<sup>4</sup> M<sup>–1</sup> s<sup>–1</sup>) for dehydration of d-mannonate,
(2) low efficiency (<i>k</i><sub>cat</sub>/<i>K</i><sub>M</sub> = 10<sup>1</sup> to 10<sup>2</sup> M<sup>–1</sup> s<sup>–1</sup>) for dehydration of d-mannonate and/or D-gluconate, and 3) no-activity with either d-mannonate
or d-gluconate (or any other acid sugar tested). Thus, the
ManD subgroup is not isofunctional and includes d-gluconate
dehydratases (GlcDs) that are divergent from the GlcDs that have been
characterized in the mandelate racemase subgroup of the ENS (Lamble
et al. FEBS Lett. 2004, 576, 133–136) (Ahmed et al. Biochem. J. 2005, 390, 529–540). These
observations signal caution for functional assignment based on sequence
homology and lay the foundation for the studies of the physiological
functions of the GlcDs and the promiscuous ManDs/GlcDs
Experimental Strategies for Functional Annotation and Metabolism Discovery: Targeted Screening of Solute Binding Proteins and Unbiased Panning of Metabolomes
The
rate at which genome sequencing data is accruing demands enhanced
methods for functional annotation and metabolism discovery. Solute
binding proteins (SBPs) facilitate the transport of the first reactant
in a metabolic pathway, thereby constraining the regions of chemical
space and the chemistries that must be considered for pathway reconstruction.
We describe high-throughput protein production and differential scanning
fluorimetry platforms, which enabled the screening of 158 SBPs against
a 189 component library specifically tailored for this class of proteins.
Like all screening efforts, this approach is limited by the practical
constraints imposed by construction of the library, i.e., we can study
only those metabolites that are known to exist and which can be made
in sufficient quantities for experimentation. To move beyond these
inherent limitations, we illustrate the promise of crystallographic-
and mass spectrometric-based approaches for the unbiased use of entire
metabolomes as screening libraries. Together, our approaches identified
40 new SBP ligands, generated experiment-based annotations for 2084
SBPs in 71 isofunctional clusters, and defined numerous metabolic
pathways, including novel catabolic pathways for the utilization of
ethanolamine as sole nitrogen source and the use of d-Ala-d-Ala as sole carbon source. These efforts begin to define an
integrated strategy for realizing the full value of amassing genome
sequence data
Experimental Strategies for Functional Annotation and Metabolism Discovery: Targeted Screening of Solute Binding Proteins and Unbiased Panning of Metabolomes
The
rate at which genome sequencing data is accruing demands enhanced
methods for functional annotation and metabolism discovery. Solute
binding proteins (SBPs) facilitate the transport of the first reactant
in a metabolic pathway, thereby constraining the regions of chemical
space and the chemistries that must be considered for pathway reconstruction.
We describe high-throughput protein production and differential scanning
fluorimetry platforms, which enabled the screening of 158 SBPs against
a 189 component library specifically tailored for this class of proteins.
Like all screening efforts, this approach is limited by the practical
constraints imposed by construction of the library, i.e., we can study
only those metabolites that are known to exist and which can be made
in sufficient quantities for experimentation. To move beyond these
inherent limitations, we illustrate the promise of crystallographic-
and mass spectrometric-based approaches for the unbiased use of entire
metabolomes as screening libraries. Together, our approaches identified
40 new SBP ligands, generated experiment-based annotations for 2084
SBPs in 71 isofunctional clusters, and defined numerous metabolic
pathways, including novel catabolic pathways for the utilization of
ethanolamine as sole nitrogen source and the use of d-Ala-d-Ala as sole carbon source. These efforts begin to define an
integrated strategy for realizing the full value of amassing genome
sequence data