Graduation Year

2009

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Molecular Medicine

Major Professor

Gloria C. Ferreira, Ph.D.

Committee Member

W. Lee Adair, Jr., Ph.D.

Committee Member

R. Kennedy Keller, Ph.D.

Committee Member

James Garey, Ph.D.

Committee Member

Huntington Potter, Ph.D.

Committee Member

Ann Smith, Ph.D.

Keywords

Heme, Pyridoxal, Pyridoxal 5' phosphate, Biotin, ALAS, AONS

Abstract

5-Aminolevulinate synthase (ALAS) and 8-amino-7-oxononanoate synthase (AONS) are two of four homodimeric members of the alpha-oxoamine synthase family of pyridoxal 5'-phosphate (PLP)-dependent enzymes. The evolutionary relationships among α-oxoamine synthases representing a broad taxonomic and phylogenetic spectrum have been examined to help identify residues that may regulate substrate specificity.

The structural plasticity of ALAS has been documented in studies of functional circularly permuted ALAS variants and the single polypeptide chain ALAS dimer (ALAS/ALAS) exhibiting a greater turnover number than wild-type ALAS. An examination of the contribution of each ALAS/ALAS active site to the enzymatic activity shows that each active site makes distinct contributions to the steady-state activity of the enzyme. Chimeric ALAS/AONS proteins exhibited an oligomeric structure with two sites having ALAS activity and two sites having AONS activity. Remarkably, the steady-state rates for both the ALAS and AONS activities were lower than that observed in the parent enzymes, while the reactivity of the ALAS sites in ALAS/AONS was similar to that of wild-type ALAS. We propose that the different contribution of each active site to the steady-state activity of ALAS/ALAS and the reduced steady-state activities of the ALAS/AONS chimera, compared to the parent enzymes, relate to different extents of conformational changes associated with product release due to the strain caused with the linking the two ALAS (or ALAS and AONS) subunits. Thus, the extensive plasticity seen in ALAS extends to another member of the α-oxoamine family, AONS.

In the α-oxoamine synthase family a conserved histidine hydrogen bonds with the phenolic oxygen of PLP and may be significant for substrate-binding, PLP-positioning, and maintaining the pKa of the imine nitrogen. The replacement of this conserved histidine, H282, with alanine in murine erythroid ALAS has multiple effects on the spectral, binding, and kinetic properties of the enzyme and supports the conclusion that H282 plays multiple roles in the enzymology of ALAS. Altogether, these results imply that amino acid H282 coordinates the movement of the pyridine ring with the reorganization of the active-site hydrogen bond network and acts as a hydrogen bond donor to the phenolic oxygen to maintain the protonated Schiff base and enhance the electron sink function of the PLP cofactor.

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