Use of Modern Molecular Techniques to Determine the Synthetic Pathway of a Novel
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Use of Modern Molecular Techniques to Determine the Synthetic Pathway of a Novel Amino Add. Most of the biosynthetic pathways described in our book were determined before the development of recombinant DNA technology and genomics, so the techniques were quite different from those that researchers would use today. Through this question, you will explore an example of the use of modern molecular techniques to investigate the pathway of synthesis of a novel amino acid, (2S)-4-amino-2-hydroxybutyrate (AHBA). AHBA is a gamma- amino acid that is a component of some aminoglycoside antibiotics, including the antibiotic but rosin. Antibiotics modified by the addition of an AHBA residue are often more resistant to inactivation by bacterial antibiotic-resistance enzymes. As a result, understanding how AHBA is synthesized and added to antibiotics is useful in the design of pharmaceuticals. In an article published in 200S, Li and coworkers describe how they determined the synthetic pathway of AHBA from glutamate. Briefly describe the chemical transformations needed to convert glutamate to AHBA. At this point, don't be concerned about the order of the reactions. Li and colleagues began by cloning the but rosin biosynthetic gene cluster from the bacterium Bacillus circulants, which makes large quantities of but rosin. They Identified five genes that are essential for the pathway, btrI, btrJ, btrK, btrO. and btrV. They cloned these genes into E. coli plasmids that allow overexpression of the genes, producing proteins with "histidine tags" fused to their amino termini to facilitate purification). The predicted amino acid sequence of the Btrl protein showed strong homology to known acyl carrier proteins. Using mass spectrometry, Li and colleagues found a molecular mass of 11.812 for the purified Btrl protein (Including the His tag). When the purified Btrl was incubated with coenzyme A and an enzyme known to attach CoA to other acyl carrier proteins, the majority molecular species had an M, of 12, 153. How would you use these data to argue that Btrl can function as an acyl carrier protein with a CoA prosthetic group? Using standard terminology, Li and coauthors called the form of the protein lacking CoA apo Btrl and the form with CoA holo-Btrl. When holo-Btrl was incubated with glutamine, ATP, and purified BtrJ protein, the holo-Btrl species of M, 12, 153 was replaced with a species of M, 12, 281, corresponding to the thioester of glutamate and holo-Btrl. Based on these data, the authors proposed the following structure for the M, 12, 281 species (gamma-glutamyl-S-Btrl): What other structure(s) is (are) consistent with the data above? Li and coauthors argued that the structure shown here (gamma-glutamyl-S-Btrl) is likely to be correct because the alpha-carboxyl group must be removed at some point in the synthetic process. Explain the chemical basis of this argument. The BtrK protein showed significant homology to PLP-dependent amino acid decarboxylases, and BtrK isolated from E. coli was found to contain tightly bound PLP. When gamma-glutamyl-S-Btrl was incubated with purified BtrK, a molecular species of M, 12.240 was produced. What is the most likely structure of this species? Interestingly, when the investigators incubated glutamate and ATP with purified Btrl, BtrJ, and BtrK, they found a molecular species of M, 12.370. What is the most likely structure of this species? Li and colleagues found that BtrO is homologous to monooxygenase enzymes that hydroxylate alkanes, using FMN as a cofactor, and BtrV is homologous to an NAD(P)H oxidoreductase. Two other genes in the cluster, btrG and btrH, probably encode enzymes that remove the gamma-glutamyl group and attach AHBA to the target antibiotic molecule. Based on these data, propose a plausible pathway for the synthesis of AHBA and its addition to the target antibiotic, include the enzymes that catalyze each step and any other substrates or cofactors needed (ATP, NAD, etc.). Reference Li, Y., Llewellyn, N.M., Giri, R., Huang, F., & Spencer, J.B. (2005) Biosynthesis of the unique amino acid side chain of but rosin: possible protective-group chemistry in an acyl carrier protein-mediated pathway. Chem. Biol. 12, 665-675.Explanation / Answer
AHBA can be formed from glutamate by gamma glutamylation of an Acyl carrier protein derived gamma aminobutyrate intermediate. This is an example of protective group synthesis.
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