Use of Modern Molecular Techniques to Determine the Synthetic Pathway of a Novel Amino Acid Most of the biosynthetic pathways described in this chapter were determined before the development of recombinant DNA technology and genomics, so the techniques were quite different from those that researchers would use today. Here we explore an example of the use of modern molecular techniques to investigate the pathway of synthesis of a novel amino acid, $(2 S)-4$ -amino- 2 -hydroxybutyrate (AHBA). The techniques mentioned here are described in various places in the book; this problem is designed to show how they can be integrated in a comprehensive study.
AHBA is a $y$ -amino acid that is a component of some aminoglycoside antibiotics, including the antibiotic butirosin. 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 $2005,$ Li and coworkers describe how they determined the synthetic pathway of AHBA from glutamate.
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(a) 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 butirosin biosynthetic gene cluster from the bacterium Bacillus circulans, which makes large quantities of butirosin. They identificd five genes that are essential for the pathway: $b t r I, b t r J, b t r K, b t r O,$ and $b t r V$. 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 (see p. 332). The predicted amino acid sequence of the BtrI protein showed strong homology to known acyl carrier proteins (see Fig. $21-5$ ). Using mass spectrometry, Li and colleagues found a molecular mass of 11,812 for the purificd BtrI protein (including the His tag). When the purificd BtrI was incubated with coenzyme A and an enzyme known to attach CoA to other acyl carrier proteins, the majority molecular species had an $M_{\mathrm{r}}$ of $12,153 .$
(b) How would you use these data to argue that BtrI 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-BtrI and the form with CoA (linked as in Fig. $21-5$ ) holo-BtrI. When holo-BtrI was incubated with glutamine, ATP, and purificd BtrJ protein, the holo-BtrI species of $M_{\mathrm{r}}$
12,153 was replaced with a species of $M_{\mathrm{r}} 12,281,$ corresponding to the thioester of glutamate and holo-Btri. Based on these data, the authors proposed the following structure for the $M_{\mathrm{r}} 12,281$ species, $\gamma$ -glutamyl-S-BtrI:
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(c) What other structure(s) is (are) consistent with the data above?
(d) Li and coauthors argued that the structure shown here ( $\gamma$ -glutamyl-S-BtrI) 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. (Hint: See Fig. $18-6,$ reaction C.) 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-BtrI was incubated with purificd BtrK, a molecular species of $M_{\mathrm{r}}$
12,240 was produced.
(c) What is the most likely structure of this species?
(f) When the investigators incubated glutamate and ATP with purified BtrI, BtrJ, and BtrK, they found a molecular species of $M_{\mathrm{r}} 12,370 .$ What is the most likely structure of this species? Hint: Remember that BtrJ can use ATP to $\gamma$ -glutamylate nucleophilic groups. Li and colleagues found that BtrO is homologous to monooxygenase enzymes (see Box
21-1) that hydroxylate alkanes, using FMN as a cofactor, and BtrV is homologous to an NAD(P)H oxidoreductase. Two other genes in the cluster, btr $G$ and $b t r H,$ probably encode enzymes that remove the $\gamma$ -glutamyl group and attach AHBA to the target antibiotic molecule.
(g) 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, ctc.).