Mechanistic Studies of 1-Deoxy-D-Xylulose-5-Phosphate Synthase from Deinococcus radiodurans.

Sumit Handa,Tyler J Spradling,Daniel R Dempsey,Justin K White,H Lee Woodcock,David J Merkler,Nanci Cook,Divya Ramamoorthy,Wayne C Guida

doi:10.21767/2471-8084.100051

Abstract

The non-mevalonate dependent (NMVA) pathway for the biosynthesis of isopentenyl pyrophosphate and dimethylallyl pyrophosphate is the sole source of these terpenoids for the production of isoprenoids in the apicomplexan parasites, in many eubacteria, and in plants. The absence of this pathway in higher organisms has opened a new platform for the development of novel antibiotics and anti-malarials. The enzyme catalyzing the first step of the NMVA pathway is 1-deoxy-D-xylulose-5-phosphate synthase (DXPS). DXPS catalyzes the thiamine pyrophosphate- and Mg (II)-dependent conjugation of pyruvate and D-glyceraldehyde-3-phosphate to form 1-deoxy-D-xylulose-5-phosphate and CO2. The kinetic mechanism of DXPS from Deinococcus radiodurans most consistent with our data is random sequential as shown using a combination of kinetic analysis and product and dead-end inhibition studies. The role of active site amino acids, identified by sequence alignment to other DXPS proteins, was probed by constructing and analyzing the catalytic efficacy of a set of targeted site-directed mutants.

Highlights

Isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) are the precursors for isoprenoids, the largest family of biologically active compounds
We have produced and characterized a series of D. radiodurans D-xylulose-5-phosphate synthase (DXPS) mutants, the selection based upon the sequence alignment of a number of DXPS enzymes and relationships to key amino acids in other Thiamine pyrophosphate (TPP)-dependent enzymes
The results of our mutation studies point to differences in the roles for Arg-423, Asp-430, and His-434 in substrate binding and catalysis for D. radiodurans DXPS relative to the equivalent amino acids in transketolase, another

Summary

Introduction

Isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) are the precursors for isoprenoids, the largest family of biologically active compounds. The isoprenoid family of molecules represents the most diverse set of biochemical entities known and are of considerable biological importance. Cholesterol (an isoprenoid) serves as a precursor to the glucocorticoids, the androgens, the mineralocorticoids, the gestagens, and estrogen [1,2]. The biomedical importance of the isoprenoids has intrigued the scientific community on numerous levels for decades, with one of the earliest questions being: “How these molecules are synthesized in the cell?” This question was answered for cholesterol by Bloch, Cornforth, Lynen, and Popják in the 1930’s through the 1960’s [3,4,5,6]. A key intermediate in the canonical pathway of cholesterol and the isoprenoid biosynthesis is mevalonate

Methods

Results

Conclusion