3.3.2 Proposed biosynthetic scheme
The combination of bioinformatic analyses and experimental results discussed thus far allow for a feasible biosynthetic route to gaburedins to be proposed. Primarily, the proposed decarboxylase function for GbnA, and the presence of a conserved GABA moiety across all of the urea natural products identified as being produced by the gbnR::aprastrain, strongly suggest that L-glutamate is the cognate GbnA substrate.
The decarboxylation product of L-glutamate would be GABA, which could react spontaneously with CO2 to yield a carbamate intermediate 108, shown in Scheme 3.2.
As this reaction would be reversible (one would expect a carbamate intermediate would readily be hydrolysed back to GABA in aqueous conditions) intermediate108could be trapped by activation with ATP, to form one of two AMP-ester intermediates, 109 or 110.
Bioinformatic analyses suggested GbnB to be an AMP-ligase and experimental evidence suggested GbnB to be an essential enzyme for gaburedin biosynthesis. Hence GbnB was proposed to adenylate either the carboxyl or the carbamoyl group of compound 108, (Scheme 3.2). Carboxyl adenylate 110 would need to undergo cyclisation to theN-carboxyanhydride111, which would then afford the gaburedins via ring opening with amino acids (this would involve nucleophilic attack at the less electrophilic carbonyl group of111, which is chemically unlikely). On the other hand, the carbamoyl adenylate 109 could react with the appropriate amino acids either directly, or via anhydride111, to form gaburedins.
Other urea-containing natural products were discussed in Section 2.3 and include syringolins and pacidamycins.163-165 Interestingly, the adenylation domain of SylC,
which has been shown to catalyse urea formation in syringolin biosynthesis, shares 39% similarity and 22% identity with GbnB.165 The mechanism for SylC-catalysed urea formation has been proposed to involve a five-membered cyclic N- carboxyanhydride 115, or a closely related species (Scheme 3.3). This anhydride is hypothesized to be formed via an analogous mechanism to that suggested for the formation of 111(Scheme 3.2). Trapping of the anhydride with an isoleucyl or valinyl acyl carrier protein thioester would lead to formation of the ureido linkage of the syringolins. As noted above, an analogous mechanism for the formation of the gaburedins via intermediate111seems unlikely.
The AMP-carbamate intermediate 109 is sufficiently reactive that it would be a short- lived species in aqueous conditions; therefore it is likely that it would readily react with Scheme 3.2–Proposed biosynthetic route to gaburedins. L-glutamate is decarboxylated to give GABA, which may proceed by one of two possible intermediates109or111, to furnish gaburedins
excess intracellular nucleophiles. Therefore, the incorporation of phenylalanine, leucine, isoleucine, valine, methionine and N-acetyl cysteine into the gaburedins A-F (80-85) may reflect the relative availability of amino acids in the cell at the time of formation of the AMP-intermediate 109, rather than specific incorporation of these amino acids by an enzyme recognising particular structural features (GbnB). In the case of gaburedin F, this could be formed via acetylation of a cysteine-containing gaburedin, or by N-acetyl cysteine attacking the AMP-ester intermediate. However, both of these routes to gaburedin F would require a poorly-nucleophilic amide nitrogen lone pair (or an ureido-nitrogen lone pair) attacking an electrophile, both of which are chemically unlikely.
The presence of such a reactive intermediate species109/111is also consistent with the observation that a vast range of different substrates are incorporated into gaburedin Scheme 3.3– Biosynthetic origin of ureido-linked dipeptides in syringolin biosynthesis as proposed by Imker and co-workers165
natural products; both L- and D- amino acids are tolerated aminoacyl moeities. The biosynthetic scheme proposed here would also account for the additional gaburedins 106and107resulting from feeding experiments with L-glutamic acid both L-glutamate and the decarboxylation product GABA can also both act as nucleophiles to react with 109/111.
Finally, the GbnC enzyme (belonging to a large family of small amino acid / peptide transporters) may be involved with export of the gaburedins into the extracellular milieu. Alternatively, GbnC may be involved in export of the AMP-carbamate intermediate 109, allowing nucleophiles in the extracellular medium to react with it to furnish gaburedins.
3.3.3 Origin of the ureidyl carbon
To probe the biosynthetic origin of the central carbon atom and GABA moieties (blue in Figure 3.1), incorporation experiments with13C-labelled precursors were carried out. If the precursor to gaburedins is glutamate, one would expect that feeding the gbnR::apra mutant with 13C-labelled L-glutamic acid would result in 13C labelled gaburedins.
Initially, to probe the origin of the ureidyl carbonyl carbon, the gbnR::apra strain was grown on SMMS enriched with [1-13C]-L-glutamic acid added to a 10 mM final concentration. If the ureidyl carbonyl is directly incorporated from the carboxylate group lost during the decarboxylation of L-glutamate to yield GABA (Scheme 3.2), then the 13C label from [1-13C]-L-glutamic acid added to the media would be expected to be retained through the biosynthesis, and therefore observed in the gaburedins extracted from this culture extract (Scheme 3.2). LC-MS analysis of the gaburedins
extracted from the gbnR::apra strain grown on SMMS with [1-13C]-L-glutamic acid revealed that there was no labelling of the gaburedins A-F (80-85) present in the culture extract (Figure 3.9). The only gaburedin extracted that contained a 13C label was the gaburedin 106 derived from attack of the carbamate intermediate by [1-13C]-L- glutamate, confirmed by the increase in the m/z values of the [M+H]+, [M+Na]+ and [M+H–C5H7NO3]+ions by 1, compared with gaburedin 106derived from unlabelled L-
glutamic acid (Figure 3.9).
Figure 3.9 – Mass spectra of gaburedins 80, 106 and 107 from metabolites extracted from the S. venezuelae gbnR mutant when grown on SMMS and SMMS enriched with 10 mM [1-13C]-L-glutamic
acid illustrating the only labelled gaburedin is gaburedin106, the product of the reaction of [1-13C]-L- glutamic acid with the proposed biosynthetic intermediate109/111
The result of this labelling experiment therefore confirms that the ureidyl carbon is not derived from L-glutamate. It was predicted that if the ureidyl carbon is derived from sodium hydrogen carbonate, that specific labelling of gaburedins would be observed when the gbnR::aprastrain was grown on SMMS enriched with 10 mM NaH13CO3 as
in the case of the ureidyl carbon present in syringolins.165 LC-MS analysis of the culture extract revealed that the mass spectra of the gaburedins observed were unchanged compared to those extracted from unmodified SMMS (Figure 3.10), suggesting that the ureidyl carbon is not derived from sodium hydrogen carbonate.
3.3.4 Origin of the conserved GABA moiety
The gbnR::apra strain was then grown on SMMS enriched with [U-13C]-L-glutamic acid, as it was hypothesised four of the 13C labels would be retained throughout the biosynthesis of the gaburedins and therefore be detected in the gaburedins extracted. This would be consistent with the previous observation that the C-1 carbon lost in the decarboxylation step is not retained in the final gaburedin natural products observed in the culture extract, but the other four carbon atoms are retained in gaburedins produced. Figure 3.10 – Mass spectra of gaburedin A (80) in metabolites extracted from theS. venezuelae gbnR mutant when grown on SMMS (top panel) and SMMS enriched with 10 mM NaH13CO
3(bottom panel),
illustrating that the13C label from NaH13CO
Figure 3.11– Biosynthetic route to gaburedins, showing the proposed incorporation of13C labels upon
feeding thegbnR::aprastrain with [U-13C]-L-glutamic acid (top panel). Mass spectra of gaburedins A80,
106and107from metabolites extracted from theS. venezuelae gbnRmutant when grown on SMMS and SMMS enriched with 10 mM [U-13C]-L-glutamic acid (bottom panel), showing retention of13C labels in
Indeed, LC-MS analysis of the gaburedins 80-85 present in the culture extract of the gbnR::apra strain on SMMS enriched with 10 mM [U-13C]-L-glutamic acid revealed that there is specific incorporation of four13C labels into all six gaburedins A-F (Figure 3.11 shows the mass spectra of gaburedin A upon feeding the gbnR::apramutant with [U-13C]-L-glutamic acid compared with feeding with 10 mM unlabelled L-glutamic acid). Although the isotope patterns in the mass spectra are complicated, the predominantm/zfor the [M+H]+species is shifted from 295.0 to 299.0 for gaburedin A 80, however the fragment ion [M+H-C5H7NO3]+ still has m/z = 166.0 as would be
expected if the 13C labels are in the conserved GABA moiety, and are therefore lost upon loss of the neutral C5H7NO3fragment.
In addition, consistent with previous data from feeding with unlabelled L-glutamic acid, the gaburedin 107 derived from GABA was also observed, again the m/z of its molecular ion being increased in value by 4 from m/z = 232.9 to 236.9, and the [M+Na]+peak increased in value fromm/z= 254.9 to 258.9.
Furthermore, the mass spectrum of the gaburedin 106 derived from L-glutamate showed an increase in itsm/z= 276.9 to 281.9 and 286.0 for the [M+H]+ion, consistent with the incorporation of four 13C labels into the conserved ureidyl-GABA moiety, in addition to five 13C labels consistent with the addition of [U-13C]-L-glutamic acid to the proposed AMP-carbamate intermediate (Figure 3.11). UHR-LC-MS analysis confirmed the molecular formulae of the ions for the unlabelled and labelled gaburedins containing13C-labels.
The relative abundance of the labelled vs unlabelled peaks in the mass spectra for gaburedins A-F 80-85, gaburedin 106 and gaburedin 107 shown in Figure 3.11 may
have been affected by the relative amounts of labelled vs unlabelled glutamate present in the media. It would be expected that the relative intensities of unlabelled:labelled peaks would be approximately 1:4 for each gaburedin, as there is 2.8 mM L-glutamic acid in the media from the casaminoacids, yet the media was supplemented with 10 mM [U-13C]-L-glutamic acid, also. The complex fragmentation patterns observed may simply reflect the complicated role of L-glutamic acid in central metabolism, and supplementing the growth media with such a large quantity (final concentration of 12.8 mM L-glutamic acid in total compared to 2.8 mM in unmodified SMMS) may have diverted metabolic flux to other pathways rather than into gaburedin biosynthesis.
3.4 Probing substrate diversity of GbnA and GbnB
3.4.1 Substrate specificity of GbnA
Contrary to the bioinformatic analysis of GbnA, the experimental evidence presented thus far implies that L-glutamate, rather than L-ornithine, L-lysine or L-arginine is utilised by GbnA as a substrate. To determine whether any gaburedin analogues could be derived from decarboxylation of L-ornithine, L-lysine or L-arginine, thegbnR::apra mutant was grown on SMMS supplemented with these amino acids. Extracellular metabolites produced after 3 days were then extracted and submitted to LC-MS analyses.
As the enzyme GbnA and its orthologues belong to the Orn/Lys/Arg decarboxylase family, extracted ion chromatograms were generated for them/z of species that would be expected if the decarboxylated form of the precursor molecules were incorporated to give gaburedin-like compounds derived from GABA analogues. For example, decarboxylation of L-ornithine would yield putrescine, L-lysine would yield
Scheme 3.4–Biosynthetic route to gaburedin analogues that would be produced if GbnA decarboxylated