Investigating the bifunctionality of cyclizing and "classical" 5-aminolevulinate synthases.

Abstract

The precursor to all tetrapyrroles is 5-aminolevulinic acid, which is made either via the condensation of glycine and succinyl-CoA catalyzed by an ALA synthase (the C4 or Shemin pathway) or by a pathway that uses glutamyl-tRNA as a precursor and involves other enzymes (the C5 pathway). Certain ALA synthases also catalyze the cyclization of ALA-CoA to form 2-amino-3-hydroxycyclopent-2-en-1-one. Organisms with synthases that possess this second activity nevertheless rely upon the C5 pathway to supply ALA for tetrapyrrole biosynthesis. The C5 N units are components of a variety of secondary metabolites. Here, we show that an ALA synthase used exclusively for tetrapyrrole biosynthesis is also capable of catalyzing the cyclization reaction, albeit at much lower efficiency than the dedicated cyclases. Two absolutely conserved serines present in all known ALA-CoA cyclases are threonines in all known ALA synthases, suggesting they could be important in distinguishing the functions of these enzymes. We found that purified mutant proteins having single and double substitutions of the conserved residues are not improved in their respective alternate activities; rather, they are worse. Protein structural modeling and amino acid sequence alignments were explored within the context of what is known about the reaction mechanisms of these two different types of enzymes to consider what other features are important for the two activities.