Directed evolution to re-adapt a co-evolved network within an enzyme.

We have previously used targeted active-site saturation mutagenesis to identify a number of transketolase single mutants that improved activity towards either glycolaldehyde (GA), or the non-natural substrate propionaldehyde (PA). Here, all attempts to recombine the singles into double mutants led to unexpected losses of specific activity towards both substrates. A typical trade-off occurred between soluble expression levels and specific activity for all single mutants, but many double mutants decreased both properties more severely suggesting a critical loss of protein stability or native folding. Statistical coupling analysis (SCA) of a large multiple sequence alignment revealed a network of nine co-evolved residues that affected all but one double mutant. Such networks maintain important functional properties such as activity, specificity, folding, stability, and solubility and may be rapidly disrupted by introducing one or more non-naturally occurring mutations. To identify variants of this network that would accept and improve upon our best D469 mutants for activity towards PA, we created a library of random single, double and triple mutants across seven of the co-evolved residues, combining our D469 variants with only naturally occurring mutations at the remaining sites. A triple mutant cluster at D469, E498 and R520 was found to behave synergistically for the specific activity towards PA. Protein expression was severely reduced by E498D and improved by R520Q, yet variants containing both mutations led to improved specific activity and enzyme expression, but with loss of solubility and the formation of inclusion bodies. D469S and R520Q combined synergistically to improve k(cat) 20-fold for PA, more than for any previous transketolase mutant. R520Q also doubled the specific activity of the previously identified D469T to create our most active transketolase mutant to date. Our results show that recombining active-site mutants obtained by saturation mutagenesis can rapidly destabilise critical networks of co-evolved residues, whereas beneficial single mutants can be retained and improved upon by randomly recombining them with natural variants at other positions in the network

Clinical-dosimetric relationship between lacrimal gland dose and ocular toxicity after intensity-modulated radiotherapy for sinonasal tumours

ObjectiveTo characterise the relationship between lacrimal gland dose and ocular toxicity among patients treated by intensity-modulated radiotherapy (IMRT) for sinonasal tumours.Methods40 patients with cancers involving the nasal cavity and paranasal sinuses were treated with IMRT to a median dose of 66.0 Gy. Toxicity was scored using the Radiation Therapy Oncology Group morbidity criteria based on conjunctivitis, corneal ulceration and keratitis. The paired lacrimal glands were contoured as organs at risk, and the mean dose, maximum dose, V10, V20 and V30 were determined. Statistical analysis was performed using logistic regression and the Akaike information criterion (AIC).ResultsThe maximum and mean dose to the ipsilateral lacrimal gland were 19.2 Gy (range, 1.4-75.4 Gy) and 14.5 Gy (range, 11.1-67.8 Gy), respectively. The mean V10, V20 and V30 values were 50%, 25% and 17%, respectively. The incidence of acute and late Grade 3+ toxicities was 23% and 19%, respectively. Based on logistic regression and AIC, the maximum dose to the ipsilateral lacrimal gland was identified as a more significant predictor of acute toxicity (AIC, 53.89) and late toxicity (AIC, 32.94) than the mean dose (AIC, 56.13 and 33.83, respectively). The V20 was identified as the most significant predictor of late toxicity (AIC, 26.81).ConclusionA dose-response relationship between maximum dose to the lacrimal gland and ocular toxicity was established. Our data suggesting a threshold relationship may be useful in establishing dosimetric guidelines for IMRT planning that may decrease the risk of acute and late lacrimal toxicities in the future.Advances in knowledgeA threshold relationship between radiation dose to the lacrimal gland and ocular toxicity was demonstrated, which may aid in treatment planning and reducing the morbidity of radiotherapy for sinonasal tumours