Search

The class 1A enzyme dihyroorotate dehydrogenase from Lactococcus lactis is examined to understand mechanism of flavin reduction. It is found that oxidation of dihyroorotate (DHO) to orotate (OA) by this enzyme is concerted, where the active site cysteine acts as a classical general base.

The study provides a detailed thermodynamic framework for electron transfer in this complex iron-sulphur flavoprotein. Their potentiometric analysis indicates that all of the redox centers in dihydroorotate dehydrogenase B (DHODB) titrate in the range of -200 to -320 mV. The detailed thermodynamic basis for electron transfer in DHODB provides a framework for transient kinetic studies of electron transfer.

The flavin reduction in dihydroorotate dehydrogenase of Escherichia coli by dihydroorotate results in a reduced flavin-orotate charge-transfer complex. The rate of reduction is pH dependent suggesting Ser175 to be the active-site base. Data indicate that quinone menadione is a good oxidizing substrate for the enzyme.

Research has been conducted on the orotidine 5'-monophosphate decarboxylase. The decarboxylation of the orotidine 5'-monophosphate has been examined.

Research was conducted to examine the importance of the unusual proline, designated Pro 131 in the Escherichia coli UPRTase, for enzyme activity. The residue was changed to an aspartate and the mutant P131d enzyme was purified to compare its catalytic properties with the properties of the wild-type protein. Results indicate that the proline in the 5-phosphoribosyl-alpha-pyrophosphate (PRPP) binding site of UPRTase has little influence in the binding of PRPP to the free enzyme.

The rate and equilibrium constants for the formation of orotidine 5'-monophosphate from orotate phosphoribosyltransferase have been studied through equilbrium binding, isotope partitioning and chemical quench studies. Phosphoribosyltransferase are nucleotide synthases that catalyze the ribose 5-phosphate group transfer during nucleotide synthesis. They are also used in the formation of nucleotide-like intermediates in tryptophan and histidine biosynthesis.

Dihydroorotate dehydrogenase (DHOD), a flavin-containing enzyme, catalyzes the oxidation of dihydroorotate (DHO) to orotate, an aromatic intermediate in the biosynthesis of pyrimidine. Cys 130 has been hypothesized to act as a base, activating DHO for hydride transfer, and results have been obtained consistent with the proposal, using chemical modification and site-directed mutagenesis.

The fold of a flexible loop region implicated in its participation in catalysis was identified in the structure of the Escherichia coli orotate phosphoribosyltransferase (OPRTase) enzyme. E. coli OPRTase is a homodimer consisting of sulfate-binding ions linked to the dimer interface near the flexible loop region. The OPRTase loop structure was found in only one of the subunits. Its association from one monomer to the active site of the adjacent monomer was mediated by bound sulfate ions.

Growth rate of carbon sources determines the protein chain and mRNA elongation of the lacZ gene. The translation elongation rate is associated with the transcription elongation rate. Growth rate dependency is detected in the synthetically fused rrnB and infB gene transcription. The elongation rate of the untranslated rrnB and RNA chain is double that of mRNA chains in lacZ and infB genes.

Escherichia coli strains W3110 and MG1655 have both been used as type strains representing the wild type strain K-12. However, previous findings have shown that these two strains have a requirement for pyrimidine. Comparisons involving W3110 and MG1655 with the rph-pyrE operon from strain HfrH showed that the W3110 rph gene contains a frameshift mutation, which results in the low expression of pyrE, which codes for orotate phosphribosyltransferase. This leads to the pyrimidine requirement phenotype.

Results of P1 transduction, cloning and sequencing of dinD fusion of E. coli reveal that the DNA damage-inducible gene dinD is identical to ordY gene in the upstream of pyrE. A DC-1 amino acid sequence of orfY matches with a part of dinD gene. The DC-1 sequence is a pseudo-attachment site for the transposon Tn7.

The catalytic function and high specificity for xanthine of Bacillus subtilis xanthine phosphoribosyltransferase (XPRTase) are examined by ligand binding studies and reaction kinetics as a function of pH with xanthine, hypoxanthine and guanine as substrates. The unique base specificity is related to a few key residues in the active site.

Crystal structures are determined for Sulfolobus solfataricus uracil phosphoribosyltransferase (UPRTase) in complex with uridine 5'-monophosphate (UMP) and with the UMP and the allosteric inhibitor CTP. A structure with UMP bound in half of the active sites is determined.

A product inhibition experiment showed that the enzyme from Escherichia coli, compared to enzyme from Lactococcus lactis A, has separate binding sites for both dihydroorotate and the electron receptor. Trypsin quickly cleaved the E coli enzyme into two fragments with 182 and 154 residues each, a reaction that reduced activity by over a hundred-fold but left all other properties unchanged. The trypsin at the E coli R182 cleavage site is positioned in a conserved site that has a serine residue.