Biochemistry


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0 Q&A 4934 Views Jan 5, 2019
D-amino acid transaminase (D-AAT) is able to synthesize both D-glutamate and D-alanine, according to the following reaction: D-alanine + α-ketoglutarate ⇌ D-glutamate + pyruvate. These two D-amino acids are essential components of the peptidoglycan layer of bacteria. In our recently published work, MSMEG_5795 from Mycobacterium smegmatis was identified as having D-amino acid transaminase (D-AAT) activity, although it has primarily been annotated as 4-amino-4-deoxychorismate lyase (ADCL). To unequivocally demonstrate D-AAT activity from MSMEG_5795 protein two coupled enzyme assays were performed in series. First, D-alanine and α-ketoglutarate were converted to D-glutamate and pyruvate by MSMEG_5795 using the D-AAT assay. Next, the products of this reaction, following removal of all protein, were used as input into an assay for glutamate racemase in which D-glutamate is converted to L-glutamate by glutamate racemase (Gallo and Knowles, 1993; Poen et al., 2016). As the only source of D-glutamate in this assay would be from the reaction of D-alanine with MSMEG_5795, positive results from this assay would confirm the D-AAT activity of MSMEG_5795 and of any enzyme tested in this manner.
0 Q&A 7799 Views Apr 5, 2018
Bacteria live in polymicrobial communities under tough competition. To persist in a specific niche many species produce toxic extracellular effectors as a strategy to interfere with the growth of nearby microbes. One of such effectors are the non-canonical D-amino acids. Here we describe a method to test the effect of D-amino acid production in fitness/survival of bacterial subpopulations within a community. Co-cultivation methods usually involve the growth of the competing bacteria in the same container. Therefore, within such mixed cultures the effect on growth caused by extracellular metabolites cannot be distinguished from direct physical interactions between species (e.g., T6SS effectors). However, this problem can be easily solved by using a filtration unit that allows free diffusion of small metabolites, like L- and D-amino acids, while keeping the different subpopulations in independent compartments.

With this method, we have demonstrated that D-arginine is a bactericide effector produced by Vibrio cholerae, which strongly influences survival of diverse microbial subpopulations. Moreover, D-arginine can be used as a cooperative instrument in mixed Vibrio communities to protect non-producing members from competing bacteria.
0 Q&A 7479 Views Jul 20, 2017
Cystinuria is a rare genetic disorder characterized by recurrent, painful kidney stones, primarily composed of cystine, the dimer of the amino acid cysteine (Sumorok and Goldfarb, 2013). Using a mouse model of cystinuria, we have recently shown that administration of drugs that increase cystine solubility in the urine can be a novel therapeutic strategy for the clinical management of the disease (Zee et al., 2017). There is a large unmet need in the field for developing new drugs for cystinuria. To that end, here we describe a simple in vitro cystine solubility assay that is amenable for screening compounds to identify potential drugs that may influence cystine solubility. The assay includes preparing a supersaturated solution of cystine, incubating this solution with drug(s) of choice, and finally using high pressure liquid chromatography–tandem mass spectrometry (HPLC-MS/MS) to quantify the amount of cystine precipitated under various conditions.
0 Q&A 11332 Views Sep 20, 2016
GABA (γ-amino butyric acid) is a biologically active four carbon non-protein amino acid found in prokaryotes and eukaryotes. Glutamate is a five carbon α-amino acid which can be converted to GABA catalyzed by the enzyme glutamate decarboxylase. In this protocol, we describe the procedure for extraction and quantification of GABA and glutamate from cells of the cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis). Apart from Synechocystis, this protocol has already been successfully tested for the cyanobacterium Nostoc punctiforme and for the green alga Tetraspora sp. CU2551. The protocol can also be used for the analyses of other primary amino acids. We have successfully employed this protocol in our studies of GABA and glutamate analyses in Synechocystis (Kanwal and Incharoensakdi, 2016).



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