Natural products and drug discovery: an evolutionary take

A question (of the nice variety) that students sometimes ask is about the relevance of natural products. The answer is a yes-and-no answer. I like to add to the usual arguments, an evolutionary take, which is two sided. A lot of secondary metabolites made by plants have been evolved to kill you (a mostly herbivore), but your liver has been evolved to be good at destroying them. For drug discovery, this has two opposite effects: the pro is a long list of antibacterial, antifungal and anticancer compounds to use or to adapt, the con is their ADME (absorption, distribution, metabolism, and excretion) properties have frustrated medchemists for decades and decades. Herein, the two faces of this coin are explored not by advocating for or against natural products, but exploring what they mean for medicinal chemistry.

Skewed mutational biases: English scrabble with Klingon tiles

Skewed mutational biases: English scrabble with Klingon tiles

In a game of Scrabble one draws seven tiles from a bag and the best results are found when a bag and the language used match. In library making with error-prone PCR one does not have that luxury, but different methods differing degrees of fairness. Mutazyme is like playing in English with a Latin tile set, while manganese Taq with equal nucleotides is like playing in English with a Klingon tile set.

In vivo shuffling via heteroduplex amplicons

Bluescreen transilluminator photo showing
different shades of green.

In a previous post I discuss the heteroduplicity of epPCR plasmids. Namely often colonies in a library will have bases that have two equal variants. The most likely cause being that the amplicons do not anneal perfect and that the transformed plasmids are actually heteroduplexes. These can either divide before being corrected or are corrected by mismatch repair.
I did a sneaky experiment to see if this could be used to make protocol to shuffle mutations between variants and got a positive result, but possibly not as effective as hoped.

The heteroduplicity of error prone PCR plasmids

A mix of wt and

mutant...

In an error prone PCR the ep-aDNA is ligated onto a plasmid backbone and transformed. When assessing the diversity from a naïve plasmid pool, something odd is seen: some bases are mutated but not to saturation. This is often just dismissed or simply overlooked, but I suspect it is actually something interesting...

Cysteine racemase: an impossible enzyme?

Cysteine racemase is an enzyme (EC 5.1.1.10) that was characterised in lysates long ago, but have never been found since. Is it a real enzyme? Can the reaction happen well? The problem is that racemising via a carbanion intermediate something with a leaving group is not an easy feat.

Biochemical reaction yield and enzyme promiscuity

Reaction yield, i.e. the molar percentage of product over substrate, is often mentioned by chemists, but never by biochemists. My guess is that many enzymes are not perfectly efficient, but have a range of reaction yields.
In The hitchhiker's guide to the galaxy a ship is hidden thanks to the "somebody else's problem" principle, namely people will ignore something problematic that isn't their problem. The reaction yield of enzymes is not something often discussed. The reason is pretty self evident: differentiating between low abundance products would be a minefield of pesky technical issues. So it is somebody else's problem.

Promiscuously hitchhiking on a pathway

In the seminal 1976 paper on enzyme evolution Roy Jensen first pointed out that the TCA cycle, the ketoadipate route to lysine, pantothenate, isoleucine, valine and leucine biosynthesis all operated via the same mechanistic steps (condensation with an acyl-CoA, rearrangement, oxidation and elimination of a carbon) and conjectured that they descent from a common primordial pathway.
Promiscuity is generally studied with a single enzyme as a model. A few paper tip-toe around it, but I am not sure that there are any that deal specifically with a pathway where the each enzyme along a pathway shows substrate ambiguity towards the promiscuous product of the previous reaction. I mentioned in another post that the branched chain amino acid pathway can produce norvaline, norleucine and homonorleucine when certain enzymes are overexpressed. Each enzyme in the pathway shows substrate ambiguity, so the whole pathway possess substrate ambiguity.
It is not a feature of the oxaloacetate-to-ketoglutarate–like pathways, but can be found in other pathways.

Pseudomonas, Ralstonia and Burkholderia: HGT buddies

Recently I was reminded of an interesting thing that intrigued me: Ralstonia and Burkholderia were formerly classed as Pseudomonas spp. and now are from different proteobacterial classes, but they share several close genes. One hypothesis that can be made is that a series of horizontal gene transfer events occurred at some point and the phenotypes of the three species became so close that they were mistakenly grouped together.

I came across the quirk while doing research for a paper from Dr. Monica Gerth and Prof. Paul Rainey:

Gerth ML, Ferla MP, Rainey PB. The origin and ecological significance of multiple branches for histidine utilization in Pseudomonas aeruginosa PAO1. Environ Microbiol. 2012 Aug;14(8):1929-40. doi: 10.1111/j.1462-2920.2011.02691.x. Epub 2012 Jan 9. PubMed PMID: 22225844.

The Ralstonia and Burkholderia are in different families within the Burkholderiales (Betaproteobacteria), while the Pseudomonas is in the Gammaproteobacteria. The taxonomic genus Burkholderia was coined when 7 Pseudomonas spp. were moved in 1993, while Ralstonia was created when two Burkholderia spp. were moved in 1996. The two are not sister genera.
In addition to the hut operon these three species are phylogenetically close to each other for many protein.

Using the Darkhorse server set to genus level detail, the top hits for Pseudomonas aeruginosa are

1. Azotobacter vinelandii, a bona fide pseudomonad, but given its own genus for silly reasons —okay, technically it is the Pseudomonas genus should be split.
2. Ralstonia metallidurans
3. Bermanella marisrubri
4. Chromohalobacter salexigens
5. Burkholderia xenovorans
6. Burkholderia thailandensis

With Pseudomonas fluorescens B. xenovorans is in third place. With Pseudomonas putida it's in second place, while R. metallidurans in fourth. While Ralstonia and Burkholderia pick each other up. The results for Ralstonia eutropha indicate that 2/3 of its large genome are from Cupriavidus taiwanensis (sister species of Burkholderia), which in reality means that there is some phylogenetic issue afoot.
Nevertheless, it does not explain the pseudomonad link, which is probably because the ancestors of Pseudomonas and of Burkholderia/Ralstonia got to know each other well and as a result today we have:

• Burkholderia spp. have two chromosomes and lots of plasmids, R. eutropha has over six and half thousand genes and pseudomonads are gene collectors too.
• They all have weird relationship with their sister genera or families.
• The seem to have similar lifestyles.
• The share lots of genes
• They were mistaken as pseudomonads morphologically.

This is just a mix of speculation and quick checks, but there is nevertheless a link. Which is not only curiosity, it finds data in mistakes from the past and potentially tells of dangers that may assail genome-concatenation studies.

Homologues and synonyms

All creatures great and small evolve, but evolution is not limited to the biological realm: languages evolve too and in a similar way.
My interest in languages stems from their ability to evolve with many similarities to enzyme evolution (and in light of not being able to spill the beans on my own research, I seem to post here a lot about words —And this is another!)
The technical words in linguistics differ from those in biology and I have yet to find an enlightening paper on the coevolution of synonyms and the parallels between language and genomes.

In enzymology, protein function and structure are not not always linked. Proteins with different functions can have similar structures having evolved from the same ancestral gene (functionally-divergent homologues) and proteins with different structures can perform the same function (functionally-convergent analogues).
In a language, words can have similar meanings, yet have different origins (synonyms) and words from the same origin can have different meanings.

New genes enter an organism via gene duplication, horizontal gene transfer or gene birth. Similarly, new words enter a language by duplication, loanwords (words of foreign origin) or onomatopoeiae.

English is a really great language for two reasons: it is my mother-tongue and it is a hybrid.
Regarding the latter reason, over a third of the words in the English dictionary were acquired from French due to the Norman invasion of 1066. The grammar and basic vocabulary remained that of Old English, whereas the advanced vocabulary was that of Old French.
A parallel to this can be drawn with the weird and wonderful bacterium Thermotoga maritima, 24% of whose genome is of archaeal origin: the replication and expression machinery is bacterial, but several metabolic routes are archaeal.

A loanword is a foreign word that is adopted in a language thanks to its real or percieved utility, whereas a horizontal gene transfer is an aquisition of a foreign gene in a genome thanks to its fitness benefits. The original spelling of loanwords are gradually lost in favour of a more compatible spelling and similarly the codon and amino acid compositions of trasferred genes also gradually change to match that of the new genome.

Often synomyms of different origins coexist, in English for a while the Old-Norse–originating window (vinauga, wind+eye), the Old-English–originating eagþyrl (eye+hole) and the Old-French–originating fenestre were coexisting, until one won. When French entered into the English language many synonyms died, but many survived in a figurative meaning, for example, enthrall litterally means to enslave, but nowadays it is used figurately, ie. capuring one's attention.
With genes the pressure to remove genes with identical function (be they paralogues, xenologues or analogues) depends on the selective pressure and in Eukaryotes they can coexist for some time.
Some words adopt alternative spellings, which can dissapear or gain different functions, such as the pairs yet and get, passed and past, and especial and special. Gene duplication does the same. In the IAD model of gene divergence, the new function comes before the amplification.

English, French and several other languages use the Latin alphabet to write words down. However, the phonetics of Latin lacks several sounds present in these and work-arounds are done. The letter h is used as a modified in English to make wh, th and ch sounds and vowel combinations allow extra vowel sounds. Old English used to be written with Anglo-saxon runes and then, with Christianity, switched over to the Latin one, bringing with it two heavily used runic letters absent in the Latin alphabet (þ and ƿ). However, as most typesetting letters were produced in France the letters died out. Despite the obvious simplicity they endow, new letters are very rarely added.
The work-around the lack of letters results in coding problems: the th group in Bath, Chatham Islands and Thailand differ, as the former is the fricative, the second is a word fusion (the H is not a modifier) and the latter is a plosive (a “violent” T). The only way to know is to know the exceptions or guess from the etymology, which can be misleading in thyme and Thames.
In protein several non-standard residues can be found. None of these use a recoded codon by itself, but require special sequences or post-transcriptional modifications, so the system differs slightly in that new letters (codons) cannot be added, but is similar in its workarounds.

Many differences exist, however.
Languages have grammar and generally authoritative bodies acting independantly of the evolution of the language (French academy).
Many suffices and prefices are highly constructive, a modularity that is hard to find in genes.
Enzymes have promiscuous function, minor non-physiological functions that can lead to new main activities: a property that is different in words. Words have multiple definitions and some are often confused. The word brothel used to mean prostitute, but it was confused with the word bordel and the meaning of the latter was bestowed on the former. Biannual traditionally means two-yearly, but its continual misuse as semestral means that the word has two discordant definitions, annulling its functionality. Genes have layers of regulation, whereas words don't. However, the metaphor could be stretch to equate grammar to regulation, but that may be wholly in the handwaving realm…