Formylglycinamide ribonucleotide synthetase from Escherichia coli: cloning, sequencing, overproduction, isolation, and characterization

Abstract

The purL gene of Escherichia coli encoding the enzyme formylglycinamidine ribonucleotide (FGAM) synthetase which catalyzes the conversion of formylglycinamide ribonucleotide (FGAR), glutamine, and MgATP to FGAM, glutamate, ADP, and Pi has been cloned and sequenced. The mature protein, as deduced by the structural gene sequence, contains 1628 amino acids and has a calculated Mr of 141,418. Comparison of the purL control region to other pur loci control regions reveals a common region of dyad symmetry which may be the binding site for the "putative" repressor protein. Construction of an overproducing strain permitted purification of the protein to homogeneity. N-Terminal sequence analysis and comparison of glutamine binding domain sequences (Ebbole & Zalkin, 1987) confirm the amino acid sequence deduced from the gene sequence. The purified protein exhibits glutaminase activity of 0.02% the normal turnover, and NH3 can replace glutamine as a nitrogen donor with a Km = 1 M and a turnover of 3 min-1 (2% glutamine turnover). The enzyme forms an isolable (1:1) complex with glutamine: t1/2 is 22 min at 4 degrees C. This isolated complex is not chemically competent to complete turnover when FGAR and ATP are added, demonstrating that ammonia and glutamine are not covalently bound as a thiohemiaminal available to complete the chemical conversion to FGAM. hydroxylamine trapping experiments indicate that glutamine is bound covalently to the enzyme as a thiol ester. Initial velocity and dead-end inhibition kinetic studies on FGAM synthetase are most consistent with a sequential mechanism in which glutamine binds followed by rapid equilibrium binding of MgATP and then FGAR. Incubation of [18O]FGAR with enzyme, ATP, and glutamine results in quantitative transfer of the 18O to Pi.

Genes of the Escherichia coli pur regulon are negatively controlled by a repressor-operator interaction

“…All three were initially identified as purR mutations by their P1 linkage to the man and pdxH loci and confirmed by complementation with purR+ plasmid pPR1002 (25 (18,24), purMN (28), purL (26), purEK (30,36), purHD (1), purC (Tiedeman et (Fig. 2).…”

Autoregulation of PurR repressor synthesis and involvement of purR in the regulation of pur B, purC, purL, purMN and guaBA expression in Escherichia coli

“…They demonstrated that a specific sequence (ACGCAAACGTTTTCTT) in the purF operator DNA was protected from DNaseI digestion by crude cell extracts of purR+ cells, but not of purR-cells. Sequences similar to the protected region (PUR box) in purF are present in the regulatory regions of purMN [4], purL [5] and purEK [3,61 of E. coli. It has been suggested that the expression of these genes is co-regulated with purF in both E. coli and Scilmonclla typhimurium [7], and also that hypoxhanthine and guanine are the corepressors in S. typhimurium [8].…”

Substrate specificity of benzamide synthetase involved in 4-hydroxy-3-nitrosobenzamide biosynthesis

“…2 Gn-ATs are classified primarily into two subfamilies, classes I and II, according to their glutaminase domains. 3 Glutaminase domains of class I Gn-ATs consist of B200 amino-acid residues and have a cysteine protease-type catalytic triad, formed by Cys, His, and Gln. In contrast, class II Gn-ATs have a glutaminase domain in which the N-terminal Cys is the only catalytic residue.…”