Mechanistic studies of the bifunctional deaminase-reductase RibD and aminoglycoside-N-acetyltransferase from Escherichia coli
de Lourdes Borba Magalhaes, Maria
MetadataShow full item record
Riboflavin is biosynthesized by most microorganisms and plants, while mammals depend entirely on the absorption of this vitamin from the diet to meet their metabolic needs. Therefore, riboflavin biosynthesis is an attractive target for drug design, since appopriate inhibitors would selectively target the microorganism with few side effects in humans. The second and third steps on the pathway are catalyzed by bifunctional proteins containing an N-terminal deaminase and a C-terminal reductase domain, encoded by the E. coli ribD gene. Besides the recent elucidation of the three-dimensional structure of RibD from E. coli and B. subtilis, no detailed enzymatic studies have been performed to date. In the present study we have studied the chemical and kinetic mechanism catalyzed by E. coli RibD. We have demonstrated that the rate of deamination exceeds the rate of reduction and the reductive ring opening occurs by a direct hydride transfer from C4- proR- NADPH to C'-1 of ribose, generating the ribityl moiety of riboflavin.;In the second part of this thesis, we have focused on the studies of aminoglycoside N-acetyltransferases. Clinical resistance to aminoglycosides is generally the result of the expression of enzymes that covalently modify the antibiotic, including N-acetylation. The aminoglycoside 3-N-acetyltransferase AAC(3)-IV from E. coli exhibits a very broad aminoglycoside specificity, causing resistance to a large number of aminoglycosides. We report here on the characterization of the substrate specificity and kinetic mechanism of the acetyl transfer reaction catalyzed by AAC(3)-IV, The kinetic mechanism was proposed to be random, based on the observed patterns of dead end inhibition and the non-Michaelis-Menten behavior of certain aminoglycoside substrates. A nanomolar bisubstrate analogue inhibitor was generated enzymatically using AAC(3)-IV, chloroacetyl-CoA and tobramycin. The compound exhibited linear competitive inhibition versus both AcCoA and tobramycin, confirming the rabdom binding of substrates to AAC(3)-IV. The chemical synthesis of other bisubstrate analogs in which the 6'-amino group of aminoglycosides was regioselectively coupled to CoA has been reported. We have tested the first generation of such inhibitors against the Salmonela enterica AAC(6')-Iy. We were able to explain the low affinity constants, as well as the unexpected inhibition patterns, based on the high affinity of the enzyme for its product: the CoA molecule. Indeed, the use of these bisubstrate analogues should provide valuable guidance for the study of other members of the N-acetyltransferase family of enzymes.