Mutagenesis of the S‐adenosyl‐L‐methionine binding site in Escherichia coli biotin synthase

Abstract

Biotin synthase catalyzes the final step of the biotin biosynthetic pathway involving the insertion of a sulfur atom into dethiobiotin. The active site of the enzyme contains the cofactor S‐adenosyl‐L‐methionine (AdoMet), the substrate dethiobiotin and two iron–sulfur clusters. The reaction sequence of biotin synthase is initiated by electron transfer through an iron–sulfur cluster into the AdoMet sulfonium, resulting in the cleavage of AdoMet into methionine and a 5′‐deoxyadenosyl radical. The radical is then used to abstract hydrogen from the dethiobiotin, allowing for the insertion of a sulfur atom. A second equivalent of AdoMet leads to another round of catalysis and the formation of a bond that completes the biotin thiophane ring. In this study, single‐point mutations of two conserved amino acids involved in the binding of AdoMet and dethiobiotin are described. The mutants have similar iron‐sulfur content to that of the wild‐type enzyme and variable binding properties pertaining to AdoMet and dethiobiotin. The mutants are unable to produce biotin, however, some are able to cleave AdoMet, producing 5′‐deoxyadenosine and a non‐biotin product. Mass spectrometry is used to analyze the alternate product providing possible evidence for a reaction intermediate where the mutant enzymes are incapable of completing the correct reaction sequence.