Biology Ph.D. Dissertations

Structure-function studies of 5-aminolevulinic acid (ALA) synthases

James Chege Kaganjo, Bowling Green State University


The essential metabolite 5-aminolevulinic acid (ALA) is a precursor in the production of tetrapyrroles and 2-amino-3-hydroxycyclopent-2-en-1-one (C5N unit), a substructure of C5N-containing polyketides. In non-plant eukaryotes and α-proteobacteria, the pyridoxal 5’-phosphate (PLP)-dependent ALA synthase enzyme catalyzes the synthesis of ALA. While an understanding as to the distinct roles of multiple ALA synthases in animals is well developed, much less is known about the presence of more than one enzyme in bacteria. The role of HemA and HemT ALA synthase isoenzymes in Rhodobacter sphaeroides was investigated by comparing the enzymatic properties of three HemAs and two HemTs from three strains: one strain has hemA and hemT genes present and both are expressed, another has both genes but hemT is not expressed, and the third strain has the hemA gene only. Although all five enzymes had similar kinetic properties, HemA enzymes were more sensitive to hemin with a 13-fold difference in Ki value compared to HemT. HemT was found to be sensitive to oxidation suggesting that the hemT gene encodes an enzyme that is more active anaerobically, which agrees well with the maximal anaerobic-dark transcription of hemT in strain 2.4.9 (Coulianos N, Kaganjo J, and JH Zeilstra-Ryalls. 2017. Mauscript in preparation). These properties indicate that the role of HemT is to supply ALA when inhibitory heme levels are elevated in the cell, which is thought to occur when cells undergo a transition from aerobic to anaerobic-dark respiration.

In addition to catalyzing the ALA synthesis reaction, bifunctional ALA synthases (cyclizing ALA synthases) in actinomycetes catalyze the cyclization of ALA-CoA to form the C5N unit. Whether other ALA synthases whose only known function is providing ALA for tetrapyrrole biosynthesis also possess this bifunctionality is not known. This question was investigated by comparing the ALA-CoA cyclization activity of R. sphaeroides strain 2.4.9 HemA and HemT enzymes relative to that of a bona fide cyclizing ALA synthase, AcaC of Streptomyces aizunensis strain NRLL-B-11277. Although both HemA and HemT had cyclization activity, it was only 10% and 6% that of AcaC respectively. However, the specific ALA synthase activities of these "classical" ALA synthases were 315-fold higher than that of the AcaC. A study of mutants in which conserved amino acid residue differences at positions 83 and 363 in each kind of enzyme were investigated revealed that they are not solely responsible for the differences in ALA synthase and ALA-CoA cyclization activities of these enzymes. Bioinformatic analyses suggest that the differences in activities could be due to differences in conformational changes as the enzymes undergo catalysis, and active site architecture.