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A Broad Spectrum Racemase in Pseudomonas putida KT2440 Plays a Key Role in Amino Acid Catabolism.

ABSTRACT: The broad-spectrum amino acid racemase (Alr) of Pseudomonas putida KT2440 preferentially interconverts the l- and d-stereoisomers of Lys and Arg. Despite conservation of broad-spectrum racemases among bacteria, little is known regarding their physiological role. Here we explore potential functional roles for Alr in P. putida KT2440. We demonstrate through cellular fractionation that Alr enzymatic activity is found in the periplasm, consistent with its putative periplasm targeting sequence. Specific activity of Alr is highest during exponential growth, and this activity corresponds with an increased accumulation of d-Lys in the growth medium. An alr gene knockout strain (?alr) was generated and used to assess potential roles for the alr gene in peptidoglycan structure, producing soluble signaling compounds, and amino acid metabolism. The stationary phase peptidoglycan structure did not differ between wild-type and ?alr strains, indicating that products resulting from Alr activity are not incorporated into peptidoglycan under these conditions. RNA-seq was used to assess differences in the transcriptome between the wild-type and ?alr strains. Genes undergoing differential expression were limited to those involved in amino acid metabolism. The ?alr strain exhibited a limited capacity for catabolism of l-Lys and l-Arg as the sole source of carbon and nitrogen. This is consistent with a predicted role for Alr in catabolism of l-Lys by virtue of its ability to convert l-Lys to d-Lys, which is further catabolized through the l-pipecolate pathway. The metabolic profiles here also implicate Alr in catabolism of l-Arg, although the pathway by which d-Arg is further catabolized is not clear at this time. Overall, data presented here describe the primary role of Alr as important for basic amino acid metabolism.

SUBMITTER: Radkov AD

PROVIDER: S-EPMC6034422 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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A Broad Spectrum Racemase in Pseudomonas putida KT2440 Plays a Key Role in Amino Acid Catabolism.

Radkov Atanas D AD Moe Luke A LA

Frontiers in microbiology 20180629

The broad-spectrum amino acid racemase (Alr) of Pseudomonas putida KT2440 preferentially interconverts the l- and d-stereoisomers of Lys and Arg. Despite conservation of broad-spectrum racemases among bacteria, little is known regarding their physiological role. Here we explore potential functional roles for Alr in P. putida KT2440. We demonstrate through cellular fractionation that Alr enzymatic activity is found in the periplasm, consistent with its putative periplasm targeting s ...[more]

PMID: 30008699

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Project description:Bacteria have evolved a wide range of chemoreceptors with different ligand specificities. Typically, chemoreceptors bind ligands with elevated specificity and ligands serve as growth substrates. However, there is a chemoreceptor family that has a broad ligand specificity including many compounds that are not of metabolic value. To advance the understanding of this family, we have used the PcaY_PP (PP2643) chemoreceptor of Pseudomonas putida KT2440 as a model. Using Isothermal Titration Calorimetry we showed here that the recombinant ligand binding domain (LBD) of PcaY_PP recognizes 17 different C6-ring containing carboxylic acids with KD values between 3.7 and 138 ?M and chemoeffector affinity correlated with the magnitude of the chemotactic response. Mutation of the pcaY_PP gene abolished chemotaxis to these compounds; phenotype that was restored following gene complementation. Growth experiments using PcaY_PP ligands as sole C-sources revealed functional relationships between their metabolic potential and affinity for the chemoreceptor. Thus, only 7 PcaY_PP ligands supported growth and their KD values correlated with the length of the bacterial lag phase. Furthermore, PcaY_PP ligands that did not support growth had significantly higher KD values than those that did. The receptor has thus binds preferentially compounds that serve as C-sources and amongst them those that rapidly promote growth. Tightest binding compounds were quinate, shikimate, 3-dehydroshikimate and protocatechuate, which are at the interception of the biosynthetic shikimate and catabolic quinate pathways. Analytical ultracentrifugation studies showed that ligand free PcaY_PP-LBD is present in a monomer-dimer equilibrium (KD = 57.5 ?M). Ligand binding caused a complete shift to the dimeric state, which appears to be a general feature of four-helix bundle LBDs. This study indicates that the metabolic potential of compounds is an important parameter in the molecular recognition by broad ligand range chemoreceptors.

Dataset Information

A Broad Spectrum Racemase in Pseudomonas putida KT2440 Plays a Key Role in Amino Acid Catabolism.

Publications

A Broad Spectrum Racemase in <i>Pseudomonas putida</i> KT2440 Plays a Key Role in Amino Acid Catabolism.

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