2149-79-3Relevant articles and documents
A substantial oxygen isotope effect at O2 in the OMP decarboxylase reaction: Mechanistic implications
Wepukhulu, Wickliffe O.,Smiley, Vanessa L.,Vemulapalli, Bhargavi,Smiley, Jeffrey A.,Phillips, Linda M.,Lee, Jeehiun K.
, p. 4533 - 4541 (2008)
Orotidine-5′-monophosphate decarboxylase (OMP decarboxylase, ODCase) catalyzes the decarboxylation of orotidine-5′-monophosphate (OMP) to uridine-5′-monophosphate (UMP). Despite extensive enzymological, structural, and computational studies, the mechanism of ODCase remains incompletely characterized. Herein, carbon kinetic isotope effects were measured for both the natural abundance substrate and a substrate mixture synthesized for the purpose of carrying out the remote double label isotope effect procedure, with O2 of the substrate as the remote position. The carbon kinetic isotope effect on enzymatic decarboxylation of this substrate mix was measured to be 1.0199 ± 0.0007, compared to the value of 1.0289 ± 0.0009 for natural abundance OMP, revealing an 18O2 isotope effect of 0.991 ± 0.001. This value equates to an intrinsic isotope effect of approximately 0.983, using a calculated commitment factor derived from previous isotope effect data. The measured 18O2 isotope effect requires a mechanism with one or more enzymatic processes, including binding and/or chemistry, that contribute to this substantial inverse isotope effect. 18O2 kinetic isotope effects were calculated for four proposed mechanisms: decarboxylation preceded by proton transfer to 1) O2; 2) O4; and 3) C5; and 4) decarboxylation without a preceding protonation step. A mechanism involving no pre-decarboxylation step does not appear to have any steps with the necessary substantial inverse 18O2 effect, thus calling into question any mechanism involving simple direct decarboxylation. Protonation at O2, O4, or C5 are all calculated to proceed with inverse 18O2 effects, and could contribute to the experimentally measured value. Recent crystal structures indicate that O2 of the substrate appears to be involved in an intricate bonding arrangement involving the substrate phosphoryl group, an enzyme Gln side chain, and a bound water molecule; this interaction likely contributes to the observed isotope effect.
Enzymatic Production of Non-Natural Nucleoside-5′-Monophosphates by a Thermostable Uracil Phosphoribosyltransferase
del Arco, Jon,Acosta, Javier,Pereira, Humberto M.,Perona, Almudena,Lokanath, Neratur K.,Kunishima, Naoki,Fernández-Lucas, Jesús
, p. 439 - 448 (2017/12/13)
The use of enzymes as biocatalysts applied to synthesis of modified nucleoside-5′-monophosphates (NMPs) is an interesting alternative to traditional multistep chemical methods which offers several advantages, such as stereo-, regio-, and enantioselectivity, simple downstream processing, and mild reaction conditions. Herein we report the recombinant expression, production, and purification of uracil phosphoribosyltransferase from Thermus themophilus HB8 (TtUPRT). The structure of TtUPRT has been determined by protein crystallography, and its substrate specificity and biochemical characteristics have been analyzed, providing new structural insights into the substrate-binding mode. Biochemical characterization of the recombinant protein indicates that the enzyme is a homotetramer, with activity and stability across a broad range of temperatures (50–80 °C), pH (5.5–9) and ionic strength (0–500 mm NaCl). Surprisingly, TtUPRT is able to recognize several 5 and 6-substituted pyrimidines as substrates. These experimental results suggest TtUPRT could be a valuable biocatalyst for the synthesis of modified NMPs.
Bis Phosphorochloridate: A Suitable Phosphorylating Agent for Nucleosides
Engels, Joachim,Krahmer, Ute
, p. 485 - 486 (2007/10/02)
-
