Molecular characterisation of a recombinant bovine glycine N–acyltransferase
Badenhorst, Christoffel Petrus Stephanus
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Conjugation of glycine to organic acids is an important detoxification mechanism. Metabolites of aspirin and industrial solvents, benzoic acid found in plant material and many endogenous metabolites are detoxified by conjugation to glycine. The enzyme responsible for glycine conjugation, glycine N-acyltransferase (GL YAT), is investigated in this study. The enzyme is also important for the management of organic acidemias which are inherited metabolic diseases. However, not all organic acids can be efficiently detoxified by GL YAT. Consequently, some organic acidemias, such as propionic acidemia, are difficult to treat. We hypothesise that a novel variant of GL YAT might be designed that can effectively detoxify propionic acid and several other organic acids. This novel GL YAT might eventually be used as a recombinant therapeutic enzyme for the treatment of organic acidemias. A thorough understanding of the mechanisms of substrate binding and catalysis by the enzyme is needed to design such a novel enzyme. This understanding is lacking at present. The first step to investigating the mechanics of substrate binding and catalysis is the development of a recombinant enzyme expression system. Amino acids in the protein can then be altered using site-directed mutagenesis, to study the importance of individual amino acid residues to enzyme function. No system for the expression of a biologically active recombinant human GLYAT has yet been developed. In a recent study in our laboratory, it was shown that bovine GL YAT could be expressed in a partially soluble and enzymatically active form, using expression at 15 °C and chaperone co-expression. The enzyme could not be investigated in detail because no tags for purification were fused to the protein. In this study the bovine GLYAT was expressed with a C-terminal histidine tag for affinity purification using the same system. It was confirmed that the recombinant bovine GLYAT was enzymatically active and it could be partially purified. Two major proteins were present after purification. The identity of the co-purifying protein is unknown. The enzyme reaction kinetics of the partially purified recombinant bovine GLYAT and of GLYAT isolated from bovine liver was determined and compared.The kinetic parameters of the two enzymes were similar and correlated with the values reported in the literature. The recombinant bovine GL YAT was used to elucidate the catalytic mechanism of the enzyme. A putative catalytic residue, E226, was identified on the basis of biochemical arguments and bioinformatic analyses. This proposed catalytic residue was mutated by means of site-directed mutagenesis. The E226Q mutant recombinant bovine GLYAT enzyme was compared to the wild type bovine GL YAT with regard to reaction kinetics and pH dependence of the reaction. The results suggested that bovine GL YAT uses the E226 residue as a general base catalyst to remove a proton from glycine in the reaction mechanism. This is the first time a mechanism for GL YAT activity has been worked out. Benzoyl-coenzyme A is a substrate of the GLYAT reaction. It is used as a reagent in GLYAT activity assays and kinetic investigations. Since this compound is very expensive, a method was adapted from the literature for the cost effective in-house synthesis of this compound. Three biosynthetic enzymes from Escherichia coli were cloned, sequenced, expressed and purified and then used for the synthesis of benzoyl-coenzyme A from benzoyl-pantetheine. The conversion from benzoylpantetheine to benzoyl-coenzyme A was stochiometric. After purification a yield higher than 75% was obtained. The benzoyl-pantetheine used was first synthesised from benzoic acid and pantethine by acylation under reducing conditions. All steps could be performed in a single tube. The method does not require purification of the benzoyl-pantetheine before use in the enzymatic synthesis of benzoyl-coenzyme A, minimising loss of material. To summarise, a recombinant bovine GLYAT with a C-terminal histidine tag was expressed and partially purified. The kinetic properties of the recombinant bovine GL YAT corresponded to the properties of GLYAT extracted from bovine liver. The recombinant bovine GLYAT was used to elucidate the catalytic mechanism of GLYAT by means of site-directed mutagenesis. This demonstrates the power of the recombinant expression system to studying the importance of specific amino acid residues. Benzoyl-coenzyme A was synthesised using a cost effective method adapted from the literature. In the future, photoaffinity labelling will be used to identify residues that constitute the substrate binding site of GL YAT and site-directed mutagenesis will then be used to investigate their function. It may then become possible to attempt to rationally design a GL YAT with altered substrate specificity.