Biomedical subjects

K A Flannigan

Publications and source records attributed to K A Flannigan.

3 recordsLinked to original sources

PubMed1990

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

The purD gene of Escherichia coli encoding the enzyme glycinamide ribonucleotide (GAR) synthetase, which catalyzes the conversion of phosphoribosylamine (PRA), glycine, and MgATP to glycinamide ribonucleotide, MgADP, and Pi, has been cloned and sequenced. The protein, as deduced by the structural gene sequence, contains 430 amino acids and has a calculated Mr of 45,945. Construction of an overproducing strain behind a lambda pL promoter allowed a 4-fold purification of the protein to homogeneity. N-Terminal sequence analysis and comparison of the sequence with those of other GAR synthetases confirm the amino acid sequence deduced from the gene sequence. Initial velocity studies and product and dead-end inhibition studies are most consistent with a sequential ordered mechanism of substrate binding and product release in which PRA binds first followed by MgATP and then glycine; Pi leaves first, followed by loss of MgADP and finally GAR. Incubation of [18O]glycine, ATP, and PRA results in quantitative transfer of the 18O to Pi. GAR synthetase is very specific for its substrate glycine.

Amino Acid Sequence↗

PubMed1990

Purine biosynthesis in Escherichia coli K12: structure and DNA sequence studies of the purHD locus.

The de novo purine biosynthetic enzymes 5-amino-4-imidazolecarboxamide-ribonucleotide (AICAR) transformylase (EC 2.1.2.3), IMP cyclohydrolase (EC 3.5.4.10) and glycineamide-ribonucleotide (GAR) synthetase (EC 2.1.2.2) are encoded by the purHD locus of Escherichia coli. The DNA sequence of this locus revealed two open reading frames encoding polypeptides of Mr 57,335 and 45,945 (GAR synthetase), respectively, that formed an operon. The DNA sequence, maxicell and complementation analyses all supported the concept that the Mr 57,335 polypeptide is the product of the purH gene and encodes a bifunctional protein containing both AICAR transformylase and IMP cyclohydrolase activities. The 5' end of the purHD mRNA was determined by primer extension mapping and contains two regions of dyad symmetry capable of forming 'hairpin' loops where the formation of the one would prevent the formation of the other but not vice versa. Regulation by the purR gene product was explained by the discovery of a purR binding site in the purHD control region.

Acyltransferases↗

PubMed1989

Reliable five-minute test strip method for identification of Streptococcus pyogenes.

A novel and rapid method (Strep Strip, Lab M) for identification of Streptococcus pyogenes was evaluated. The method combines the established test for pyroglutamyl aminopeptidase (PYR) with a rapid chromogenic test for beta-glucosidase on a paper strip. The test was evaluated with 274 clinical isolates and 237 culture collection isolates of beta-haemolytic streptococci. Streptococcus pyogenes was identified with 100% specificity. Six isolates identified as Lancefield group A and which might therefore be assumed to be Streptococcus pyogenes were shown by the test strip method not to be this species. The beta-glucosidase test on the test strip allowed differentiation between enterococci and Streptococcus pyogenes (both PYR positive) in all cases. The test strip method represents an economical and accurate method for identification of Streptococcus pyogenes in clinical material.

Aminopeptidases↗