Active site lysines in orotate phosphoribosyltransferase.

Grubmeyer, Charles; Segura, Edy; Dorfman, Ryan

doi:10.1016/s0021-9258(20)80728-5

Cited by 24 publications

(37 citation statements)

References 20 publications

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“…30,38,[40][41][42] Another shared feature of Type I PRTases is the catalytic role of conserved lysine residues (Fig. 8, bold) 43,44 involved in the binding of a highly charged Mg 2+ :PRPP complex and anionic form of OA at pH 8.0.…”

Section: Mass Spectrometry Identification and Molecular Mass Determin...mentioning

confidence: 99%

“…The k cat value for MtOPRT demonstrates low catalytic efficiency in comparison to P. falciparum, 14 S. cerevisiae 23 and Homo sapiens 27 homologues (Table 2). A possible explanation is that active site lysine residues that play catalytic roles (Lys26 and Lys73 in S. typhimurium) 43,44,48 are replaced with Ser26 and Gly72 in MtOPRT, thereby reducing the catalytic constant of the latter. Notwithstanding, site-directed mutagenesis efforts should be pursued to lend or not support to this proposal.…”

Section: Comparison Of Steady-state Kinetic Parametersmentioning

confidence: 99%

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Molecular, kinetic and thermodynamic characterization of Mycobacterium tuberculosisorotate phosphoribosyltransferase

et al. 2012

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Tuberculosis (TB) is a chronic infectious disease caused mainly by Mycobacterium tuberculosis. The worldwide emergence of drug-resistant strains, the increasing number of infected patients among immune compromised populations, and the large number of latent infected individuals that are reservoir to the disease have underscored the urgent need of new strategies to treat TB. The nucleotide metabolism pathways provide promising molecular targets for the development of novel drugs against active TB and may, hopefully, also be effective against latent forms of the pathogen. The orotate phosphoribosyltransferase (OPRT) enzyme of the de novo pyrimidine synthesis pathway catalyzes the reversible phosphoribosyl transfer from 5'-phospho-α-D-ribose 1'-diphosphate (PRPP) to orotic acid (OA), forming pyrophosphate and orotidine 5'-monophosphate (OMP). Here we describe cloning and characterization of pyrE-encoded protein of M. tuberculosis H37Rv strain as a homodimeric functional OPRT enzyme. The M. tuberculosis OPRT true kinetic constants for forward reaction and product inhibition results suggest a Mono-Iso Ordered Bi-Bi kinetic mechanism, which has not been previously described for this enzyme family. Absence of detection of half reaction and isothermal titration calorimetry (ITC) data support the proposed mechanism. ITC data also provided thermodynamic signatures of non-covalent interactions between substrate/product and M. tuberculosis OPRT. These data provide a solid foundation on which to base target-based rational design of anti-TB agents and should inform us how to better design inhibitors of M. tuberculosis OPRT.

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Section: Mass Spectrometry Identification and Molecular Mass Determin...mentioning

confidence: 99%

Section: Comparison Of Steady-state Kinetic Parametersmentioning

confidence: 99%

Molecular, kinetic and thermodynamic characterization of Mycobacterium tuberculosisorotate phosphoribosyltransferase

et al. 2012

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“…In the middle variant, the catalyst loop of chain B was not attracted to chain A, but that of chain A was attracted to chain B. Although the detailed reaction mechanism is under debate, the interaction between catalyst loops and another subunit is postulated to be important for PRPP binding and catalytic activity. − The RMSF value of the attracted catalyst loop in chain A was lower than that in chain B. In Simp-2, each catalyst loop was attracted to the other chain.…”

Section: Resultsmentioning

confidence: 99%

Molecular Dynamics Simulations for Three-Dimensional Structures of Orotate Phosphoribosyltransferases Constructed from a Simplified Amino Acid Set

et al. 2020

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Proteins of modern terrestrial organisms are composed of nearly 20 amino acids; however, the amino acid sets of primitive organisms may have contained fewer than 20 amino acids. Furthermore, the full set of 20 amino acids is not required by some proteins to encode their function. Indeed, simplified variants of Escherichia coli (E. coli) orotate phosphoribosyltransferase (OPRTase) constructed by Akanuma et al. and composed of a limited amino acid set exhibit significant catalytic activity for the growth of E. coli. However, its structural details are currently unclear. Here, we predict the structures of simplified variants of OPRTase using molecular dynamics (MD) simulations and evaluate the accuracy of the MD simulations for simplified proteins. The three-dimensional structure of the wild-type was largely maintained in the simplified variants, but differences in the catalyst loop and C-terminal helix were observed. These results are considered sufficient to elucidate the differences in catalytic activity between the wild-type and simplified OPRTase variants. Thus, using MD simulations to make structural predictions appears to be a useful strategy when investigating non-wild-type proteins composed of reduced amino acid sets.

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“…typhimurium and in Escherichia coli) , is that it closes the active site of the adjacent subunit establishing hydrogen bond interactions with the PRPP substrate. This feature has been proposed to be essential for PRPP binding and catalysis. ,, The OPRTase structures reveal that the enzyme undergoes different conformational changes depending on the complex formed (apoenzyme, primary complex with OA, binary product complex with OMP, or ternary complex with OA, Mg 2+ , and PRPP). While the apoenzyme is similar between two microorganisms (S.…”

Section: Introductionmentioning

confidence: 99%

“…This feature has been proposed to be essential for PRPP binding and catalysis. 18,20,23 The OPRTase structures reveal that the enzyme undergoes different conformational changes depending on the complex formed (apoenzyme, primary complex with OA, binary product complex with OMP, or ternary complex with OA, Mg 2+ , and PRPP). While the apoenzyme is similar between two microorganisms (S. cerevisiae and S. typhimurium), large conformational changes take place in those structures that contain the substrates.…”

Section: Introductionmentioning

confidence: 99%

Elucidating the Catalytic Reaction Mechanism of Orotate Phosphoribosyltransferase by Means of X-ray Crystallography and Computational Simulations

et al. 2020

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Active site lysines in orotate phosphoribosyltransferase.

Cited by 24 publications

References 20 publications

Molecular, kinetic and thermodynamic characterization of Mycobacterium tuberculosisorotate phosphoribosyltransferase

Molecular, kinetic and thermodynamic characterization of Mycobacterium tuberculosisorotate phosphoribosyltransferase

Molecular Dynamics Simulations for Three-Dimensional Structures of Orotate Phosphoribosyltransferases Constructed from a Simplified Amino Acid Set

Elucidating the Catalytic Reaction Mechanism of Orotate Phosphoribosyltransferase by Means of X-ray Crystallography and Computational Simulations

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