Graduation Year

2012

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Chemistry

Major Professor

David J. Merkler

Keywords

1-deoxy-D-xylulose-5-phosphate Synthase, D. radiodurans, non mevalonate pathway, P. vivax

Abstract

Isoprenoids are the largest family of biologically active compounds, synthesized by five carbon subunits namely isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). For long time the mevalonate-dependent (MVA) pathway has been considered as the sole source of IPP and DMAPP, until recently a new non-mevalonte dependent (NMVA) pathway was discovered. This new pathway utilizes entirely different set of enzymes for isoprenoids synthesis and don't have any homologues in humans. NMVA pathway is the only source of isoprenoids for certain eubacteria, parasite and plants. Absence of the NMVA pathway in higher organisms has opened a new platform for the development of novel antibiotics and antimalarials.

1-deoxy-D-xylulose-5-phosphate synthase (DXS), the first enzyme in NMVA pathway has been reported as the rate limiting enzyme in the synthesis of IPP and DMAPP and has been the center of interest for inhibitor development. Reaction mechanism of thiamine pyrophosphate (TPP) and Mg2+ dependent DXS enzyme has been studied in this report. Using steady state kinetics analysis, product inhibition and dead end inhibitor, the mechanism of substrate (pyruvate and D-glyceraldehyde-3-phosphate) addition was studied. Due to different domain organization in DXS as compared to theother TPP dependent enzyme, the mechanism of addition was found to be random sequential rather than ping-pong mechanism.

Based on bioinformatics tool and in vitro studies it has been established that NMVA exists in all the plasmodium species, thus making the enzymes involved in NMVA as an alluring target for new antimalarial drugs. All the plasmodium species and other member of the phylum apicomplexa harbor apicoplast an organelle which is homologous to the chloroplast of plants and algae. All the enzymes from NMVA pathway translocate to apicoplast from nucleus through a secretory pathway using signaling and transit peptide. In this study DXS from P. vivax has been cloned and expressed in E. coli using genomic DNA and codon optimized synthetic DNA as a source. Expression of full length DXS with signal and transit peptide as well as mature protein without these peptide using serial deletion has been studied. Kinetic parameters of P.vivax DXS have been calculated and found to be comparable to the DXS from other species.

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