On the Evolution of Catalysis: The changing kinetics of core metabolic enzymes

enzymes;evolution;mutagenesis;enzyme evolution;(ba)8-barrels;TIM-barrel;HisA;ASR;Ancestral sequence reconstruction;MurA;AroA;TrpF;substrate assisted catalysis

Newton, Matilda Skye ; Patrick, Wayne

University of Otago

关键词: enzymes;    evolution;    mutagenesis;    enzyme evolution;    (ba)8-barrels;    TIM-barrel;    HisA;    ASR;    Ancestral sequence reconstruction;    MurA;    AroA;    TrpF;    substrate assisted catalysis;   

Others  :  https://ourarchive.otago.ac.nz/bitstream/10523/5803/1/NewtonMatildaS2015PhD.pdf

美国|英语

来源: Otago University Research Archive

【 摘 要 】

Enzymes are biological catalysts that are essential to life: they are largely very efficient and exquisitely specialised to their substrate. They are, however, predicted to have evolved from simple promiscuous catalysts (able to catalyse multiple reactions on multiple substrates). The goal of this thesis was to explore the multiple models of evolution with three model enzymes.In a complementary model for enzyme evolution, it is hypothesised that some ancient enzymes may have exhibited higher catalytic rates than their extant descendants. This model was investigated through the reconstruction of core bacterial enzymes AroA (aromatic amino acid biosynthesis) and MurA (peptidoglycan biosynthesis), from the common ancestor of modern Streptoccocci species. The ~300 million year old ancestral enzyme conformed to the model, with 20-fold higher activity than MurA enzymes in modern Streptococci.Several models for enzyme evolution, both primordial and contemporary, require a multifunctional precursor enzyme as a starting point. This was the case in a previous study, in which model enzyme HisA (histidine biosynthesis) from Salmonella enterica was evolved to acquire novel activity towards the TrpF (tryptophan biosynthesis) substrate (Näsvall et al., 2012). In the current study, the variant enzymes representing a mutational trajectory between HisA and TrpF were kinetically characterised and the structure-function link identified for many causative mutations. A three-amino acid duplication was key for establishing the novel TrpF function, altering the induced fit mechanism of the enzyme and repositioning the general acid side chain. Other amino acid substitutions improved novel activity and substrate specificity by excluding the HisA substrate.The S. enterica HisA active site and that of the variant specialised to TrpF activity were characterised through site-directed mutagenesis and kinetic assays. It was found that the new TrpF enzyme employed two different catalytic mechanisms. In addition to an entirely enzyme-based mechanism (as in HisA), the enzyme also employed substrate-assisted catalysis, whereby a functional group of the substrate contributed to catalysis. Such primitive mechanisms might – like multifunctional enzymes – be common intermediates during the evolution of enzymatic function.The requirements for TrpF function in the S. enterica HisA active site were further probed through site-directed mutagenesis to find the relative contribution of different residues to function. A duplicated arginine residue was important to alter the orientation of the active site aspartate to optimise its role as a general acid. A library generated through site-saturation mutagenesis in the active site of a bifunctional HisA variant was selected for improved TrpF function and yielded an enzyme with 13-fold improved activity and absolute substrate specificity.This study has demonstrated the ruggedness of the extended HisA landscape and how highly connected the enzyme is with other functions. The elegant complexity of enzymatic function and its evolution is demonstrated on many different fronts.