Structure and Function of Cyanobacterial DHDPS and DHDPR

Janni B. Christensen,Santosh Panjikar,T. P. Soares da Costa,Pierre Faou,F. Grant Pearce,Matthew A. Perugini

doi:10.1038/srep37111

Abstract

Lysine biosynthesis in bacteria and plants commences with a condensation reaction catalysed by dihydrodipicolinate synthase (DHDPS) followed by a reduction reaction catalysed by dihydrodipicolinate reductase (DHDPR). Interestingly, both DHDPS and DHDPR exist as different oligomeric forms in bacteria and plants. DHDPS is primarily a homotetramer in all species, but the architecture of the tetramer differs across kingdoms. DHDPR also exists as a tetramer in bacteria, but has recently been reported to be dimeric in plants. This study aimed to characterise for the first time the structure and function of DHDPS and DHDPR from cyanobacteria, which is an evolutionary important phylum that evolved at the divergence point between bacteria and plants. We cloned, expressed and purified DHDPS and DHDPR from the cyanobacterium Anabaena variabilis. The recombinant enzymes were shown to be folded by circular dichroism spectroscopy, enzymatically active employing the quantitative DHDPS-DHDPR coupled assay, and form tetramers in solution using analytical ultracentrifugation. Crystal structures of DHDPS and DHDPR from A. variabilis were determined at 1.92 Å and 2.83 Å, respectively, and show that both enzymes adopt the canonical bacterial tetrameric architecture. These studies indicate that the quaternary structure of bacterial and plant DHDPS and DHDPR diverged after cyanobacteria evolved.

Highlights

The structure of dihydrodipicolinate reductase (DHDPR) has been determined from several bacterial species, including Corynebacterium glutamicum[31], E. coli[32,33], M. tuberculosis[34,35] and S. aureus[36]
We show that Anabaena variabilis (Av)-DHDPS and Av-DHDPR both adopt the canonical bacterial structures, suggesting that the point of quaternary structural divergence between the bacterial and plants enzymes occurred after cyanobacteria evolved
Av-DHDPR were expressed in E. coli as His-tagged constructs and purified to >9 8% homogeneity using immobilised metal affinity chromatography (IMAC) (Fig. 2)

Summary

Introduction

The structure of DHDPR has been determined from several bacterial species, including Corynebacterium glutamicum[31], E. coli[32,33], M. tuberculosis[34,35] and S. aureus[36]. These studies show that the enzyme exists as a homotetramer with a unique quaternary architecture. Given that endosymbiotic theory suggests that the chloroplasts of plants were derived from the symbiosis of separate single bacterial cells[38], we were interested in characterising the structure and function of DHDPS and DHDPR from the model cyanobacterial species, Anabaena variabilis (Av)[39]. We show that Av-DHDPS and Av-DHDPR both adopt the canonical bacterial structures, suggesting that the point of quaternary structural divergence between the bacterial and plants enzymes occurred after cyanobacteria evolved

Objectives

Methods

Results

Conclusion