Discovery of a novel l -lyxonate degradation pathway in Pseudomonas aeruginosa PAO1

Salehe Ghasempur, Subramaniam Eswaramoorthy, Brandan S. Hillerich, Ronald D. Seidel, Subramanyam Swaminathan, Steven C. Almo, John A. Gerlt

Research output: Contribution to journalArticle

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Abstract

The l-lyxonate dehydratase (LyxD) in vitro enzymatic activity and in vivo metabolic function were assigned to members of an isofunctional family within the mandelate racemase (MR) subgroup of the enolase superfamily. This study combined in vitro and in vivo data to confirm that the dehydration of l-lyxonate is the biological role of the members of this family. In vitro kinetic experiments revealed catalytic efficiencies of ∼104 M -1 s-1 as previously observed for members of other families in the MR subgroup. Growth studies revealed that l-lyxonate is a carbon source for Pseudomonas aeruginosa 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 Labrenzia aggregata IAM 12614 with Mg2+ 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) 7β-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.

Original languageEnglish (US)
Pages (from-to)3357-3366
Number of pages10
JournalBiochemistry
Volume53
Issue number20
DOIs
StatePublished - May 27 2014

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Hydro-Lyases
Pseudomonas aeruginosa
mandelate racemase
Degradation
Dehydration
Phosphopyruvate Hydratase
Genes
Gene encoding
Citric Acid Cycle
Sequence Alignment
Catalytic Domain
Oxidoreductases
Substitution reactions
Carbon
Cells
X-Rays
X rays
Polymerase Chain Reaction
Kinetics
Enzymes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Ghasempur, S., Eswaramoorthy, S., Hillerich, B. S., Seidel, R. D., Swaminathan, S., Almo, S. C., & Gerlt, J. A. (2014). Discovery of a novel l -lyxonate degradation pathway in Pseudomonas aeruginosa PAO1. Biochemistry, 53(20), 3357-3366. https://doi.org/10.1021/bi5004298

Discovery of a novel l -lyxonate degradation pathway in Pseudomonas aeruginosa PAO1. / Ghasempur, Salehe; Eswaramoorthy, Subramaniam; Hillerich, Brandan S.; Seidel, Ronald D.; Swaminathan, Subramanyam; Almo, Steven C.; Gerlt, John A.

In: Biochemistry, Vol. 53, No. 20, 27.05.2014, p. 3357-3366.

Research output: Contribution to journalArticle

Ghasempur, S, Eswaramoorthy, S, Hillerich, BS, Seidel, RD, Swaminathan, S, Almo, SC & Gerlt, JA 2014, 'Discovery of a novel l -lyxonate degradation pathway in Pseudomonas aeruginosa PAO1', Biochemistry, vol. 53, no. 20, pp. 3357-3366. https://doi.org/10.1021/bi5004298
Ghasempur S, Eswaramoorthy S, Hillerich BS, Seidel RD, Swaminathan S, Almo SC et al. Discovery of a novel l -lyxonate degradation pathway in Pseudomonas aeruginosa PAO1. Biochemistry. 2014 May 27;53(20):3357-3366. https://doi.org/10.1021/bi5004298
Ghasempur, Salehe ; Eswaramoorthy, Subramaniam ; Hillerich, Brandan S. ; Seidel, Ronald D. ; Swaminathan, Subramanyam ; Almo, Steven C. ; Gerlt, John A. / Discovery of a novel l -lyxonate degradation pathway in Pseudomonas aeruginosa PAO1. In: Biochemistry. 2014 ; Vol. 53, No. 20. pp. 3357-3366.
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abstract = "The l-lyxonate dehydratase (LyxD) in vitro enzymatic activity and in vivo metabolic function were assigned to members of an isofunctional family within the mandelate racemase (MR) subgroup of the enolase superfamily. This study combined in vitro and in vivo data to confirm that the dehydration of l-lyxonate is the biological role of the members of this family. In vitro kinetic experiments revealed catalytic efficiencies of ∼104 M -1 s-1 as previously observed for members of other families in the MR subgroup. Growth studies revealed that l-lyxonate is a carbon source for Pseudomonas aeruginosa 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 Labrenzia aggregata IAM 12614 with Mg2+ 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) 7β-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.",
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