RESUMO
The uses of 3-formylbenzoic acid and 4-formylbenzoic acid as molecular probes along with previous and new transketolase mutants revealed the factors governing the rate of reaction between transketolase and aromatic aldehydes. The novel α,α-dihydroxyketones were produced at 15 to 30-fold higher yields and up to 250-fold higher specific activities with D469T TK when compared to those obtained for benzaldehyde.
Assuntos
Ácido Benzoico/química , Ácido Benzoico/metabolismo , Engenharia de Proteínas/métodos , Transcetolase/genética , Transcetolase/metabolismo , Domínio Catalítico , Escherichia coli/enzimologia , Ligação de Hidrogênio , Cinética , Simulação de Acoplamento Molecular , Mutação , Eletricidade Estática , Transcetolase/químicaRESUMO
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.
Assuntos
Evolução Molecular Direcionada/métodos , Mutagênese Sítio-Dirigida/métodos , Proteínas Recombinantes/metabolismo , Transcetolase/metabolismo , Acetaldeído/análogos & derivados , Acetaldeído/metabolismo , Aldeídos/metabolismo , Domínio Catalítico/genética , Biblioteca Gênica , Cinética , Modelos Moleculares , Mutação , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Estereoisomerismo , Especificidade por Substrato , Transcetolase/química , Transcetolase/genética , Transcetolase/isolamento & purificaçãoRESUMO
Transketolase mutants previously identified for use with the non-phosphorylated aldehyde propanal have been explored with a series of linear and cyclic aliphatic aldehydes, and excellent stereoselectivities observed.
