The Salmonella typhimurium nadC gene: Sequence determination by use of Mud-P22 and purification of quinolinate phosphoribosyltransferase

Abstract

The Salmonella typhimurium nadC gene and its product, quinolinic acid phosphoribosyltransferase (QAPRTase), were characterized at the molecular and biochemical levels. Fusions of Mud-lac elements isolated in the nadC gene were converted to Mud-P22 insertions. Starting with six original Mud-lac fusions, the entire sequence of the nadC gene was readily obtained. The sequence shows a long open reading frame with two potential initiator methionines, one of which is preceded by the Shine-Dalgarno sequence GGAG-7- nucleotide-ATG. The protein predicted from this second open reading frame is 297 residues in length. The nadC gene was subcloned into a T7-based expression system, allowing for facile purification of the QAPRTase (EC 2.4.2.19) protein to homogeneity. Upon gel filtration, the protein gave an M(r) of 72,000, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a subunit M(r) of 35,000. Automated Edman degradation of several tryptic peptides confirmed the amino acid sequence predicted from the DNA sequence. Chromatography of the apparently homogeneous enzyme on reverse-phase high- performance liquid chromatography resolved two protein species. One of these species failed to give an amino-terminal sequence, while the other yielded the amino-terminal sequence predicted by the second open reading frame and lacked the initiator methionine. The mass of the mature protein, predicted from its DNA sequence, was 32,428 Da. Electrospray mass spectrometry gave masses of 32,501 and 32,581 Da for the two peptides. Steady-state kinetics on the purified QAPRTase indicated K(m) values of 32 μM for 5-phosphoribosyl- 1-pyrophosphate and 20 μM for quinolinate. V(max) was 0.9 U/mg, similar to values reported for this enzyme by other sources.