Functional Evidence for the Critical Amino-Terminal Conserved Domain and Key Amino Acids of Arabidopsis 4-HYDROXY-3-METHYLBUT-2-ENYL DIPHOSPHATE REDUCTASE    

Abstract

The plant 4-HYDROXY-3-METHYLBUT-2-ENYL DIPHOSPHATE REDUCTASE (HDR) catalyzes the last step of the methylerythritol phosphate pathway to synthesize isopentenyl diphosphate and its allyl isomer dimethylallyl diphosphate, which are common precursors for the synthesis of plastid isoprenoids. The Arabidopsis (Arabidopsis thaliana) genomic HDR transgene-induced gene-silencing lines are albino, variegated, or pale green, confirming that HDR is essential for plants. We used Escherichia coli isoprenoid synthesis H (Protein Data Bank code 3F7T) as a template for homology modeling to identify key amino acids of Arabidopsis HDR. The predicted model reveals that cysteine (Cys)-122, Cys-213, and Cys-350 are involved in iron-sulfur cluster formation and that histidine (His)-152, His-241, glutamate (Glu)-242, Glu-243, threonine (Thr)-244, Thr-312, serine-379, and asparagine-381 are related to substrate binding or catalysis. Glu-242 and Thr-244 are conserved only in cyanobacteria, green algae, and land plants, whereas the other key amino acids are absolutely conserved from bacteria to plants. We used sitedirected mutagenesis and complementation assay to confirm that these amino acids, except His-152 and His-241, were critical for Arabidopsis HDR function. Furthermore, the Arabidopsis HDR contains an extra amino-terminal domain following the transit peptide that is highly conserved from cyanobacteria, and green algae to land plants but not existing in the other bacteria. We demonstrated that the amino-terminal conserved domain was essential for Arabidopsis and cyanobacterial HDR function. Further analysis of conserved amino acids in the amino-terminal conserved domain revealed that the tyrosine-72 residue was critical for Arabidopsis HDR. These results suggest that the structure and reaction mechanism of HDR evolution have become specific for oxygen-evolving photosynthesis organisms and that HDR probably evolved independently in cyanobacteria versus other prokaryotes.

The amino-terminal conserved domain of 4-hydroxy-3-methylbut-2-enyl diphosphate reductase is critical for its function in oxygen-evolving photosynthetic organisms

“…21 Unexpectedly, the IspH domain of the Arabidopsis HDR corresponding to the full-length IspH of E. coli failed to complement the E. coli ispH mutant. 21 Nonetheless, several key amino acids involved in iron-sulfur formation, substrate binding or catalytic reaction in E. coli IspH were conserved in Arabidopsis HDR. Homology modeling and substrate docking of the IspH domain of the Arabidopsis HDR revealed that these key residues were also located in the central active site.…”

“…Homology modeling and substrate docking of the IspH domain of the Arabidopsis HDR revealed that these key residues were also located in the central active site. 21 In addition to the conserved key amino acids, the Arabidopsis HDR contains several critical residues that are absent in the bacterial enzyme. 21 These results implicate that the reaction mechanism of plant HDR may be different from that of E. coli IspH.…”

“…21 In addition to the conserved key amino acids, the Arabidopsis HDR contains several critical residues that are absent in the bacterial enzyme. 21 These results implicate that the reaction mechanism of plant HDR may be different from that of E. coli IspH. The Arabidopsis MEP pathway enzymes are predicted to localize to the plastid.…”

“…2B and C). 21 The NCD of Arabidopsis HDR includes amino acids 56 to 110, which is followed by the IspH domain. We used protein BLAST (http:// blast.st-va.ncbi.nlm.nih.gov/Blast.cgi) to search the database and did not find any domain or motif that was similar to the NCD.…”

“…12,13 In Arabidopsis thaliana, the hdr-1 knockout mutant is albino lethal and the HDR transgene-induced genesilencing lines are albino, pale green, or variegated. 18,21 The Arabidopsis HDR was able to complement the E. coli ispH mutant indicating that the function of this enzyme is conserved from bacteria to plants. 18,22 The structure and reaction mechanisms of IspH have been extensively studied in E. coli and Aquifex aeolicus.…”