Greetings everyone.
During my master's research, I focused on exploring the potential of a specific bacterial strain to produce antibacterial compounds. To achieve this, I used the technique of liquid-liquid fractionation (Extraction) using butanol for the bacterial culture broth. Then, I subjected the supernatant to freeze-drying and further dissolved a portion of the butanol crude extract in methanol for analysis using GC-MS. The results revealed the presence of two secondary metabolite compounds, notably beta-carboline and cyclo-l-proline-l-leucine.
I investigated the genes and enzymes of the bacteria, and it appears that the genes and enzymes that synthesize beta-carboline and related compounds were not present in the bacteria. I have the genomic sequence date of the bacteria. I have searched the genome database to identify any genes or enzymes associated with the production of beta-carboline. Unfortunately, no such gene or enzyme seems to be directly related to the synthesis of beta-carboline in this bacterium. Also, my investigations regarding the McbB enzyme (which is an enzyme that works for the production of beta-carboline) have unfortunately provided no evidence of its presence in my bacterial strain.
So my question Is there an alternative methodology or approach by which I could clarify the mechanisms used by this bacterial strain to produce these compounds? For instance, the synthesis of beta-carboline usually involves the enzymatic action of tryptophan decarboxylase, which catalyzes the conversion of tryptophan to tryptamine. However, my bacterial strain seemingly lacks this specific enzyme. I am hoping that if any practical strategies or methodologies exist, I will try them as a first step to finding some answers for the synthetic pathway.
I sincerely appreciate any insights or directions you can provide.
Did you use standards for those compounds? If the peaks were automatically annotated you must view the annotations with caution.
Did you do a blast search for enzymes with that activity? Have you checked the BRENDA database for alternative enzymes? As beta-carboline is a highly conjugated ring system you probably can assay for production from precursors using absorption/RAMAN spectroscopy so would just need a crude non-denatured protein extract.
Tryptamine can also form from other oxidative processes which need not be enzymatic. This means cytochrome p450s and SAM radical enzymes could also be involved. Assuming a dopamine decarboxylase or other aromatic-L-amino acid decarboxylase with broad substrate range is not responsible.
Cyclo-l-proline-l-leucine is a cyclic dipeptide that can come from non-enzymatic autocatalysis by the N-terminal amine of proteins. Similar to N-terminal glycines having an N-terminal proline on proteins makes them prone to shedding cyclic dipeptides especially if the amino acid pair are repeated several times at the N-terminus.
Dear Mr. Robert Adolf Brinzer, I BLASTed those genes from those bacteria that produce Beta-carboline against my bacterial strain, but there were no similarities between them; the highest percentage was 23%. Also, regarding the BRENDA database, I couldn't find any alternative enzymes.
Recently, I sent the Beta-carboline standard to the GC-MS, but still I didn't get the result back. I don't know if I answered your first question which is (Did you use standards for those compounds?
With divergent enzymes you can still get functional conservation with only 16% homology. Alternatively your species might have a novel enzyme that performs the same function.
You will need to run the standard in the same run as one of your samples. Then you will have confidence in the annotation.
I guess your doubt is related to Intra-Cellular metabolites (I guess you have chosen secondary metabolite compounds instead). You can have a look at my recent publication, which was published in RSC Molecular Omics recently. It may drive you to some extent in your research focus.
https://pubs.rsc.org/en/Content/ArticleLanding/2023/MO/D3MO00141E
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