A comparison of functionally divergent forms of the purine biosynthesis enzyme pure

Kelly L Sullivan, Purdue University

Abstract

Purines are the basic building block for essential biomolecules such as DNA, RNA, NADH, CoA, and several essential vitamin cofactors. Most organisms have the ability to synthesize purines through the de novo purine biosynthesis pathway. The de novo purine biosynthesis pathway contains a rare metabolic divergence within the eukaryotes. Animals convert CO2 and aminoimidazole ribonucleotide (AIR) to carboxyaminoimidazole ribonucleotide (CAIR) using the enzyme aminoimidazole ribonucleotide carboxylase (PurE2), while other eukaryotes, archaea, and bacteria use two enzymes, N 5-carboxyaminoimidazole ribonucleotide synthetase (PurK) and N5-carboxyaminoimidazole ribonucleotide (NCAIR) mutase, PurE (PurE1). PurE catalyzes formation of the sole carbon-carbon bond formed during purine biosynthesis. Both PurE classes are proposed to generate a carbon-carbon bond using similar chemistry. We can monitor the carbon-carbon bond forming reaction in real-time using changes in intrinsic fluorescence that report upon electrostatic changes within the active site. To simplify fluorescence responses, we generated mutants of PurE1 from Acetobacter aceti (AaPurE1-W34F,W165F) and PurE2 from Treponema denticola (TdPurE2-F79W) that have a single tryptophan fluorophore, which responds to changes upon ligand addition as assessed by fluorescence titrations. Surprisingly, a Tyr residue, with ambiguous electron density in a 1.35 Å AaPurE1-W34F,W165F structure, contributes to fluorescence. Both PurE1 and PurE2 have similar structures and active sites. A universally conserved serine is the sole polar contact with the substrate aminoimidazole ring and any PurE side chain. Serine donates a hydrogen bond to the aminoimidazole N3 but may flip polarity in PurE2 at low pH. We generated a series of serine mutants in AaPurE1 and TdPurE2, which confirm a hydrogen-bonding role for serine, as the mutants with the highest detectable activity are able to donate (or accept, at low pH) a hydrogen bond. Serine is more important for PurE2 function and its role appears to be different between the two classes. The PurE2 active site contains a strictly conserved, class-specific lysine. Due to the difficult task of acquiring CO2 from solution, the lysine was proposed to form a carboxylated intermediate in PurE2 catalysis. We generated the TdPurE2-K41R mutant, which retained both AIR carboxylase and CAIR decarboxylase activity, indicating that formation of a lysine-carbamate is not an obligatory intermediate for PurE2 function. PurE2 catalyzes the formation of CAIR from AIR and CO2 without any detectable side reaction. Functional complementation assays and a crystal structure both indicated the "inactive" TdPurE2-H40N mutant may destroy AIR, CAIR, or both. We demonstrated that this "inactive" mutant does indeed destroy AIR in a slow O2-dependent oxygenase activity reaction. Due to the unusual divergence in purine biosynthesis, PurE1 is a potential antimicrobial target; however, no enzyme activity assay is suitable for high throughput screening. We developed a simple chemical quench that fixes the PurE1 substrate/product ratio for 24 h, as assessed by the Bratton-Marshall assay (BMA) for diazotizable amines. The ZnSO4 stopping reagent is proposed to chelate CAIR, allowing later analysis of this acid-labile product by BMA or a high throughput screening methods.

Degree

Ph.D.

Advisors

Kappock, Purdue University.

Subject Area

Biochemistry

COinS