[@moderators note: this email was conveyed to us from @Agauger by @Wayne_Rossiter]
@Art Hunt has posted a deceptive rebuttal to my description of the Nasvall paper. He lightly glosses over some major technical feats that were required to get the experiment to go. If either of you is willing, could you make these points for me?
- Doug and I have always granted that it is possible to recruit a protein to a new function for which it already has a low level of activity, in other words, to recruit a promiscuous enzyme to one of its minor reactions. This is likely to be the case if the chemistry of the reaction (the kinds of bonds broken and formed and their relative positions and energy requirements) and/or the substrates are similar. THIS WE HAVE ALWAYS SAID IS POSSIBLE. HisA and TrpF have been classic cases of promiscuous enzymes for a while. See below.
Jürgens C, Strom A, Wegener D, Hettwer S, Willmans M and Sterner R (2000) Directed evolution of a (βα)8-barrel enzyme to catalyze related reactions in two different metabolic pathways - PMC. Proc Natl Acad Sci 97, 9925-9930
In this paper they converted the HisA enzyme (from Thermotoga) to carry out TrpF function by directed evolution on limiting trp medium. They recovered 3 mutant strains that could grow slowly (after several days). 2 strains were identical and had four amino acid changes (4 base changes). The other strain had 3 amino acid changes (3 base changes). When all seven changes were tested individually by site directed mutagenesis, only one had a measurable effect on TrpF function [Asp127Val (A380T)]. Kcat/Km of converted enzyme was 10-5 lower than wild type. All 3 aa changes improved function four fold.
The chemistry for HisA and TrpF are the same, but the substrates have different R groups (see below). Conversion results in loss of original function, and poor new function. The original substrate ProFar for HisA is quite large-
The new substrate PRA is much smaller, but has many structural similarities.
- When Art says they used evolution to create the trpF hisA mutants he left out a bit. Here is what it took (from the Methods and Materials of the Nasvall paper) to make the double mutant they started with. To say it happened by evolution is a half-truth at best. If this is evolution then all of genetics is evolution, and geneticists should just sit back and relax and let evolution do the work. This required a sophisticated set-up, and follow through. What you see below is just the set up.
Deletion of the trpF and hisA genes
In Salmonella enterica and many other bacteria, the trpF gene is fused to the trpC gene, resulting in a bifunctional fusion protein (TrpCF). Hereafter we refer to the 3’ part of the trpCF gene (encoding aminoacids 257-452 of the fusion protein) as trpF. The hisA and trpF genes were replaced with FLP-recombinase target (FRT) site flanked kanamycin resistance (KanR ) cassettes derived from plasmid pKD4, using the lambda-red recombineering functions from plasmid pKD46 (24). The following primers were used for PCR amplification of the resistance cassettes: Sal_hisA_del_Fwd: cgcggcgggcgcacagttgctgaaaaacttcctggagatgtaatGTGTAGGCTGGAGCTGCTTC Sal_hisA_del_Rev: accatcacgaacatccagacacggaattatacgttttgccagCATATGAATATCCTCCTTAG Sal_trpF_del_Fwd: gacgatcttaacgccgccgtccgtcgcgtgctgcttgccgaaaattaatGTGTAGGCTGGAGCTGCTTC G Sal_trpCF_del_Rev2: catgccgccgaattcaccaaagtaggggttgagaagtgttgtCATATGAATATCCTCCTTAGTTCC The kanamycin cassettes in the resulting mutants, ΔhisA::kan and ΔtrpF::kan, were used as selectable markers for strain constructions, but were later removed by expression of the FLP recombinase from plasmid pCP20 (26) to generate the ΔhisA::FRT and ΔtrpF::FRT mutations.
Selection for bifunctional HisA mutants
We plated several independent cultures of a ΔtrpF::FRT, hisO1242 strain (DA15428) on M9+glycerol+histidine plates to select any mutant that could produce tryptophan without a TrpF enzyme. We included histidine in the medium because previous studies have demonstrated that mutations that confer TrpF activity to Thermotoga maritima HisA results in loss of HisA activity (27). The expression of the his operon (including hisA) is regulated by an attenuation mechanism that regulates the amount of read-through of a transcriptional terminator before the first structural gene of the operon according to the availability of charged histidinyl-tRNA (28). As this results in very low expression of hisA in medium containing histidine, we included a mutation (hisO1242) (29) which removes the transcriptional terminator, thereby leading to derepressed transcription of the his operon even in the presence of histidine. This selection resulted in very few mutants (an estimated mutation frequency of less than 10-11 / cfu). All of the isolated mutants were auxotrophic for histidine, but based on growth on M9+glycerol+histidine plates they fell in two different classes. One class was relatively fast-growing, forming colonies in 2-3 days while the other class grew more slowly, forming colonies in 4-5 days. Sequencing hisA in six independent clones from the faster growing class revealed two very similar mutations; three mutants had a duplication of nt. 37-45 and three had a duplication of nt. 36-44, resulting in duplication of the same three amino acids (V13, V14 and R15). For the rest of the experiments we have used a mutant with duplication of nt. 37-45 (GTGGTGCGT), and refer to it as hisA(dup13-15). Sequencing hisA in five independent isolates of the slower growing class of mutants revealed that they all have the same CUG (Leu) to CGG (Arg) mutation at codon 169 (L169R). As all of the isolated mutants had completely lost the original hisA activity we performed a second selection, starting with hisA(dup13-15) and hisA(L169R) mutants and selecting for growth in the absence of both histidine and tryptophan. As histidine was omitted from the medium, this selection did not require the hisO1242 mutation. Selections starting with hisA(dup13-15) resulted in relatively high frequencies of mutants (ranging between 3×107 /cfu and 6×107 /cfu in different experiments). Sequencing hisA in 18 independent mutants revealed two different mutations (in addition to the dup13-15 mutation), 15 of the mutants had a mutation resulting in a Gly to Asp substitution at amino acid 11 (G11D), while three had an Asp to Gly substitution at amino acid 10 (D10G). The same selection starting with the hisA(Leu169R) mutant also resulted in His+, Trp+ mutants (approximately 1x10-8 /cfu), but none of the tested mutants had any additional mutation in hisA. To map the mutations, a pool of approximately 20,000 random Tn10dTet insertions was generated in one of the mutants (DA16721). A phage P22 lysate grown on the transposon pool was used to transduce the parent strain (DA16608), selecting tetracycline resistance and histidine prototrophy (His+ phenotype). We found four different Tn10dTet insertions that were genetically linked to the mutation that caused the His+ phenotype. Seven more independent mutants were tested for cotransduction between one of the isolated Tn10 insertions (miaB::Tn10dTet) and the mutation that caused the His+ phenotype. All mutations were linked to the same location. Sequencing outwards from the Tn10s revealed that they were close to the metT tRNA operon. Sequencing the metT operon revealed that all eight tested mutants had a mutation in either glnX (3 mutants) or glnV (5 mutants), converting a tRNAGln to a missense suppressor tRNA with an anticodon complementary to CGG (Arg) codons. Thus, a fraction of the HisA molecules will have a L169Q substitution instead of L169R, apparently restoring at least some of the original activity. The mutation in glnX is referred to as glnXCCG (where CCG is the anticodon) in the text.
NOW HERE’S THE IRONIC THING. NASVALL ET AL DEMONSTRATED THAT PROMISCUOUS ENZYMES CAN BE SHIFTED TO A NEW FUNCTION USING MUTATION AND SELECTION JUDICIOUSLY APPLIED, AND THAT DUPLICATION AND DIVERGENCE CAN LEAD TO THE SEPARATION OF THE TWO FUNCTIONS INTO SEPARATE GENES. YOU START WITH HIS A, YOU MAKE HIS A TRP F, THEN YOU SEE IF YOU CAN GET THE GENES TO DUPLICATE AND DIVERGE. AND THEY DO! WAS IT AN EVOLUTIONARY PROCESS? IT WAS A GENETIC TOUR DE FORCE, AN EVOLUTIONARY PROCESS DESIGNED AND GUIDED BY 4 GENETICISTS. ONE OF THEM THE BEST IN HIS FIELD.
So why was I shouting? Because this is ludicrous. Evolutionary biologists are using a perfect example of evolution by design as their argument against Michael Behe. I am also yelling because apparently Art Hunt doesn’t understand anything about bacterial genetics, and how much work that would be, and he doesn’t understand anything I have written about the limits of protein evolution, or he hasn’t read it, or he is misrepresenting it. This HisA Trp F conversion is something we would say could happen. If it can be reached within a few mutations, i.e. in a genetic selection like here, then, hey, it potentially could evolve. Without investigator interference? Probably not. But potentially. If the need was great. And apparently has in different species of bacteria.
Stop with the misrepresentations already. @Art Hunt You have people reading you who don’t know the material and depend on you for the truth. So don’t twist it or play word games. Whether or not it is plausible trpF and HisA could have evolved one from the other, the fact is the Nasvall paper was an exercise in intelligently designed directed evolution. Not evolution.