Question

Use of Modern Molecular Techniques to Determine the Synthetic Pathway of a Novel Amino Acid.

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 γ-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.

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 identified five genes that are essential for the pathway: btrl, 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 BtrI 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 BtrI protein (including the His tag). When the purified BtrI was incubated with coenzyme A and an enzyme known to attach CoA to other acyl carrier proteins, the majority molecular species had an Mr 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 holo-BtrI. When holo-BtrI was incubated with glutamine, ATP, and purified BtrJ protein, the holo-BtrI species of Mr 12,153 was replaced with a species of Mr 12,281, corresponding to the thioester of glutamate and holo-BtrI. Based on these data, the authors proposed the following structure for the Mr 12,281 species (γ-glutamyl-S-BtrI):

c) What other structure(s) is (are) consistent with the data above?

d) Li and coauthors argued that the structure shown here (γ-glutamyl-S-BtrI) is likely to be correct because the α-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 γ-glutamyl-S-BtrI was incubated with purified BtrK, a molecular species of Mr 12,240 was produced.

e) What is the most likely structure of this species?

f) Interestingly, when the investigators incubated glutamate and ATP with purified BtrI, BtrJ, and BtrK, they found a molecular species of Mr 12,370. What is the most likely structure of this species? Hint: Remember that BtrJ can use ATP to γ-glutamylate nucleophilic groups.

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 γ-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, etc.).

Answer 1

a) The $\alpha$-carboxyl group is removed and an -OH (hydroxyl group) is added to the $\gamma$-carbon.

b)

• BtrI has sequence homology with acyl carrier proteins.
• The molecular weight of BtrI increases when incubated under conditions in which Coenzyme A could be added to the protein.
• Adding CoA to a Serine residue would replace an —OH (formula weight (FW) 17) with a 4'-phosphopantetheine group.
• This group has the formula C₁₁H₂N₂O₇PS(FW 356).
• Thus,

  11,182-17+356=12,151

  Note:-Which is very close to the observed M_r of 12,153

c) Thioester can combine with the $\alpha$-carboxyl group.

d)

• For removing the $\alpha$-carboxyl group of an amino acid, the carboxyl group must be free.
• Furthermore, it is difficult to imagine a decarboxylation reaction starting with a carboxyl group in its thioester form.

e)

12,240-12,281= 41

which is close to the M_r of CO₂ (44)

• We know that BtrK is probably a decarboxylase, its most likely structure is the decarboxylated form:

  

0 HN Btrl DECARBOXY

f)

12,370-12,240=130

• Glutamic Acid(C₅H₉NO₄; Mr 147), minus the -OH (hydroxyl group), FW 17 removed in the glutamylation reaction, leaves a glutamyl group of FW 130; thus,

• $\gamma$-glutamylating the molecule above would add 130 to its Mr.

• BtrJ is capable of $\gamma$-glutamylating other substrates, so it may $\gamma$-glutamylate the structure above.

• The most likely site for this is the free amino group, giving the following structure:

  

0 To NH3 Btr

g)

NH CO2 BtrJ Btrk O ATP ADP +Btr BtrI Glutamate ATP ADP H3 N BtrJ Biosynthesis of the BtrI chain of butirosin O2 H2O 0 0 Biro NHa S FMNH2 FMN Btrl BtrV NAD NADH Antibiotic 0 OH NH NHa Glutamate +Btr Antibiotic BtrI

(Semisynthetic addition of AHBA to clinically valuable aminoglycoside antibiotics has been shown to improve their pharmacological properties and to prevent their deactivation by a number of aminoglycoside-modifying enzymes involved in bacterial resistance)