Differential Effects of a Mutation on the Normal and Promiscuous Activities of Orthologs: Implications for Natural and Directed Evolution

Neutral drift occurring over millions or billions of years results in substantial sequence divergence among enzymes that catalyze the same reaction. Although natural selection maintains the primary activity of orthologous enzymes, there is, by definition, no selective pressure to maintain physiologically irrelevant promiscuous activities. Thus, the levels and the evolvabilities of promiscuous activities may vary among orthologous enzymes. Consistent with this expectation, we have found that the levels of a promiscuous activity in nine gamma-glutamyl phosphate reductase (ProA) orthologs vary by about 50-fold. Remarkably, a single amino acid change from Glu to Ala near the active site appeared to be critical for improvement of the promiscuous activity in every ortholog. The effects of this change varied dramatically. The improvement in the promiscuous activity varied from 50- to 770-fold, and, importantly, was not correlated with the initial level of the promiscuous activity. The decrease in the original activity varied from 190- to 2,100-fold. These results suggest that evolution of a novel enzyme may be possible in some microbes, but not in others. Further, these results underscore the importance of using multiple orthologs as starting points for directed evolution of novel enzyme activities.

This construction results in incorporation of Met-Gly 2 -Ser-His 6 -Gly-Met-Ala-Ser before the initial Met of ProA. For cloning into pETcoco-2, the sequences encoding the tagged ProA enzymes were amplified from the corresponding pTrcHis constructs using the following primers: forward: 5' CAG CCT GAT ACA GAT TAA ATC AGA GCG GCC GCA TCG 3'; reverse : 5' CGA TGC GGC CGC TCT GAT TTA ATC TGT ATC AGG CTG 3'. The amplified fragments were then digested with NheI and NotI for 5 hr at 37 ºC. The resulting fragments were ligated into pETcoco-2, which had been linearized by digestion with NheI and NotI, using DNA ligase for 20 min at 16 °C.
Generation of competent cells. Five mL cultures of ΔargC::kan ΔproA::cat (DE3) cells were grown overnight at 37 °C in LB containing 50 µg/mL kanamycin. The next morning, the cells were harvested by centrifugation at 9,500 x g for 5 min at 4 ºC. The cell pellet was resuspended in 100 µL of LB. Five µL of this cell suspension was inoculated into 500 mL of LB containing 50 µg/mL kanamycin. The cells were grown at 37 °C until the OD 600 reached 0.6. The cultures were incubated on ice for 20 min prior to centrifugation at 3800xg for 15 min at 4 °C. The cells were washed with 500 mL of 10% glycerol. The pellet was resuspended in 50 mL of 10% glycerol and centrifuged at 3800 x g for 15 min at 4 °C. The pellet was then resuspended in 5 mL of 10% glycerol and centrifuged at 1,900 x g for 15 min at 4 °C. The cell pellet was resuspended in 1 mL of 10% glycerol. Fifty µL aliquots were flash frozen in liquid nitrogen and stored at -80

°C.
Purification of ProA enzymes. pTrcHis plasmids encoding proA alleles were introduced into competent ΔargC::kan ΔproA::cat (DE3) cells by electroporation and the transformants were spread onto LB plates containing 100 µg/mL ampicillin. After growth, a single colony was inoculated into 5 mL of LB containing 100 µg/mL ampicillin and the cells were grown with shaking for 14 hrs at 37 °C. The cells were harvested by centrifugation at 9,500 x g for 5 min.
The cell pellet was resuspended in 1 mL LB. A 100 µL aliquot of the cell suspension was inoculated into 1 L of LB containing 100 µg/mL ampicillin. IPTG was added to a concentration of 1 mM when the OD 600 was 0.7 and the culture was grown with shaking for 14 hrs at 37 ºC.
The cells were harvested by centrifugation at 3800 x g for 15 min at 4 °C. The cell pellet was resuspended in lysis buffer (50 mM sodium phosphate, pH 8.0, 10 mM imidazole, 300 mM sodium chloride, 20 mM DTT) containing 10% glycerol (2 mg cells per mL of buffer) and stored at -80 °C. The suspended cells were lysed by two passes through a French press at 12000 psi at enzymes were purified as described in the Ni-NTA Purification System Handbook (Invitrogen).
The supernatant was loaded onto a 12 cm x 2 cm glass column containing 8-10 mL of Ni-NTA agarose (Invitrogen) that had been pre-equilibrated with lysis buffer. ProA. We added a large amount of E. coli ProA (15 µM) to a solution of NAGSA or GSA in 100 mM potassium phosphate, pH 7.6, containing 1 mM NADP + , in 1 mL. The absorbance at 340 nm due to formation of NADPH exhibited a burst followed by a linear phase. The magnitude of the burst was proportional to the total amount of GSA and P5C, whereas the slope of the linear phase was constant, regardless of the amount of GSA and P5C. We conclude that the burst represents consumption of the free aldehyde and hydrated form of the substrate, which we assume are in rapid equilibrium, while the linear phase represents the slower rate at which the P5C ring opens to form GSA. The magnitude of the burst, which typically represented 1-2% of the total concentration of GSA+P5C, was measured before each set of kinetic assays.
NAGSA and GSA dehydrogenase activities were measured by monitoring the appearance of NADPH at 340 nm in reaction mixtures containing 100 mM potassium phosphate, pH 7.6, 1 mM NADP + , varying concentrations of NAGSA or GSA, and catalytic amounts of ProA or ProA*.
All kinetic measurements were done at room temperature. Apparent values of K M based upon the total concentration of GSA+hydrate+P5C were adjusted based upon the concentration of GSA+hydrate measured as described above.
Purification of N-succinyldiaminopimelate aminotransferase/acetylornithine transamine (ArgD). E. coli argD was cloned into pET-21d in order to add a sequence encoding an N-terminal His 6 -tag and the resulting plasmid was introduced into electrocompetent E. coli DH5α cells (New England Biolabs) according to the manufacturers protocol. Transformants were selected on LB plates containing 100 µg/mL of ampicillin. A single colony from the plate was inoculated into 5 mL LB containing ampicillin (100 µg/mL) and the culture was grown overnight with shaking at 37 °C. Plasmid DNA was purified using the QiaPrep Spin Miniprep protocol (Qiagen). The purified plasmid was introduced into 50 µL of 10β cells (New England Biolabs).
The cells were allowed to recover for 1 hr at 37 °C in 1 mL of SOC medium (New England Biolabs). A 50 µL aliquot was then spread onto a plate of LB agar containing 50 µg/mL ampicillin. After overnight growth at 37 ºC, a single colony was used to inoculate 5 mL of LB containing 100 µg/mL ampicillin. The cells were grown overnight at 37 °C with shaking. The following morning, the cells were harvested by centrifugation at 4 °C for 15 min at 1900 x g.
The cell pellet was resuspended in 100 µL of LB, and a 50 µL aliquot was inoculated into 500 mL of LB containing 100 µg/mL ampicillin. The cells were grown until the OD 600 was 0.6-0.8, at which time IPTG was added to a final concentration of 1 mM. Cell growth was continued for an additional 3 hrs. The cells were harvested by centrifugation at 3800xg for 15 min at 4 °C. ArgD was purified using the protocol described above for purification of ProA.
Synthesis of N-acetylglutamate 5-semialdehyde. NAGSA was synthesized enzymatically using N-succinyldiaminopimelate aminotransferase/acetylornithine transaminase (ArgD) in a 300 mL reaction mixture containing 20 mM potassium phosphate, pH 8.5, 100 mM N-acetyl ornithine, 100 mM α-ketoglutarate, 0.01 mM pyridoxal-5'-phosphate, and 50-100 mg ArgD. After incubation for 5 hrs at 37 ºC, the reaction mixture was loaded at room temperature onto a 500 Generation of proA libraries. proA orthologs were amplified from the pTrcHisB plasmids into which they had been cloned using error-prone PCR with Mutazyme II by the following amplification protocol: Step 1, 95 °C for 2 min; Step 2, 95 °C for 30 s; Step 3, 50 °C for 30 s; Step 4, 72 °C for 1 min 30 s; Step 5, repeat steps 2-4 30 times; Step 6, 72 °C for 5 min. The PCR products were digested with NheI, BamHI and DpnI overnight at 37 °C and then purified by gel extraction prior to ligation into pTrcHisB that had been linearized by digestion with Nhe1 and BamHI. Ligation was carried out at 10 ºC overnight. The libraries were introduced into electrocompetent 10-β cells. The transformants were incubated in 1 mL SOC medium (New England Biolabs) at 37 ºC with shaking for one hour prior to plating 200 µL aliquots onto LB agar containing amplicillin (100 µg/mL). Tens of thousands of colonies from each plate were recovered in LB medium and plasmids were isolated from each sample. Each library was introduced into ΔargC::kan ΔproA1cat cells by electroporation. After the transformants were allowed to recover in LB at 37 ºC for one hour with shaking, the cells were recovered, washed twice with PBS and resuspended in 200 µL PBS. A 1 µL aliquot was spread onto agar plates containing LB and ampicillin (100 µg/mL); in each case, more than 10 4 colonies grew. The remaining cells were spread onto agar plates containing M9/glucose and 1 mM proline.
Plasmids were isolated from several colonies that grew on the M9/glucose/proline plates and the inserted proA genes were sequenced.