Radkov Atanas D, Moe Luke A
Department of Plant and Soil Sciences, College of Agriculture, University of Kentucky, Lexington, KY, United States.
Front Microbiol. 2018 Jun 29;9:1343. doi: 10.3389/fmicb.2018.01343. eCollection 2018.
The broad-spectrum amino acid racemase (Alr) of 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 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 gene knockout strain (Δ) was generated and used to assess potential roles for the gene in peptidoglycan structure, producing soluble signaling compounds, and amino acid metabolism. The stationary phase peptidoglycan structure did not differ between wild-type and Δ 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 Δ strains. Genes undergoing differential expression were limited to those involved in amino acid metabolism. The Δ 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.
KT2440的广谱氨基酸消旋酶（Alr）优先催化赖氨酸（Lys）和精氨酸（Arg）的L型和D型立体异构体相互转化。尽管细菌中广谱消旋酶具有保守性，但其生理作用却鲜为人知。在此，我们探究了Alr在KT2440中的潜在功能作用。我们通过细胞分级分离证明，Alr酶活性存在于周质中，这与其假定的周质靶向序列一致。Alr的比活性在指数生长期最高，且该活性与生长培养基中D - Lys积累的增加相对应。构建了一个基因敲除菌株（Δ），并用于评估该基因在肽聚糖结构、产生可溶性信号化合物和氨基酸代谢中的潜在作用。野生型菌株和Δ菌株在稳定期的肽聚糖结构没有差异，这表明在这些条件下，Alr活性产生的产物未掺入肽聚糖中。RNA测序用于评估野生型菌株和Δ菌株之间转录组的差异。差异表达的基因仅限于参与氨基酸代谢的那些基因。Δ菌株作为唯一碳源和氮源对L - Lys和L - Arg的分解代谢能力有限。这与Alr在L - Lys分解代谢中的预测作用一致，因为它能够将L - Lys转化为D - Lys，后者通过L - 哌啶酸途径进一步分解代谢。此处的代谢谱也表明Alr参与L - Arg的分解代谢，尽管此时D - Arg进一步分解代谢的途径尚不清楚。总体而言，本文提供的数据描述了Alr作为对碱性氨基酸代谢很重要的主要作用。
