Characterization of OrfY in the PurL gene cluster of Acetobacter aceti
FGAM synthetase, or PurL, catalyzes the fourth step of the de novo purine biosynthetic pathway where N-formylglycinamide ribonucleotide (FGAR), ATP, and glutamine yield N-formylglycinamidine ribonucleotide (FGAM), ADP, Pi, and glutamate. Two forms of PurL have been observed: one large, found in Gram-negative bacteria and eukaryotes; and the other, small, found in Gram-positive bacteria and archaea. Large PurL is a single gene product that is functional as a monomer whereas small PurL forms a αβ2γ heterotetramer with two other gene products: PurS and PurQ, which are analogous to the N-terminal and glutaminase domains of large PurL, respectively. Regardless of quaternary structure, all FGAM synthetases are thought to use ATP to O-phosphorylate the FGAR formamide, which is then attacked by ammonia to give FGAM. The acetic acid bacterium Acetobacter aceti, and closely related α-proteobacteria, differ from other Gram-negatives in that they contain purSQL genes. The structural genes for the PurSQL complex (AaFS) are clustered into an apparent operon, which also contains an open reading frame (orf) that we designate orf Y and two genes (bolA and grxD) that have key roles in the assembly of iron-sulfur clusters. OrfY has a narrow species distribution: it is found in most acetic acid bacteria and several closely related Gram-negative bacteria. OrfY possesses four conserved cysteines and an N-terminal export sequence typical of periplasmic or exported proteins. Given this unusual set of features, and the likelihood of coupled transcription-translation in the purSQYL-bolA-grxD operon, we speculate that OrfY might be an additional subunit for PurSQL, perhaps required for function in the acidic cytoplasm of acetic acid bacteria; a participant in iron-sulfur cluster assembly, which is essential for the production of the first purine pathway enzyme PurF and therefore purine biosynthesis; or as a regulatory factor. As a first step in understanding the role of OrfY, we sought to establish the functional form of the A. aceti enzyme to determine if orf Y encodes a heretofore unknown fourth subunit of the small PurL complex. This study focused on construct generation of various purine biosynthetic genes, purification of the proteins they encode, and preliminary work on gene complementation, the operon, and growth of A. aceti in minimal media. Functional complementation studies in an Escherichia coli ΔpurL strain have shown that active AaFS is likely composed of PurS, PurQ, and PurL, although the stoichiometry of the subunits is still unknown, thus indicating that OrfY is likely not a subunit of the AaFS complex. The presence of orfY in the gene cluster affected recombinant PurQ expression and caused a delay in growth in the E. coli ΔpurL strain. Recombinant OrfY was successfully purified from E. coli and mass spectrometry analysis revealed that OrfY was processed and exported.
Kappock, Purdue University.
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