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Journal of Bacteriology, December 2004, p. 8156-8158, Vol. 186, No. 23
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.23.8156-8158.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Type II Isopentenyl Diphosphate Isomerase from Synechocystis sp. Strain PCC 6803

Sam J. Barkley, Shrivallabh B. Desai, and C. Dale Poulter^*

Department of Chemistry, University of Utah, Salt Lake City, Utah

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Open reading frame sll1556 in the cyanobacterium Synechocystis sp. strain 6803 encodes a putative type II isopentenyl diphosphate (IPP) isomerase. The His₆-tagged protein was produced in Escherichia coli and purified by Ni²⁺ chromatography. The homotetrameric enzyme required NADPH, flavin mononucleotide, and Mg²⁺ for activity; K_m^IPP was 52 µM, and k_cat^IPP was 0.23 s^–1.

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The isomerization of isopentenyl diphosphate (IPP) to dimethylallyl diphosphate (DMAPP) is an essential reaction in the mevalonate (MVA) pathway to isoprenoids (13) and, although not essential, probably serves to balance the IPP and DMAPP pools in the methylerythritol phosphate (MEP) route (9). Two types of IPP isomerase are known. The type I enzyme, found in Eucarya and some of the Bacteria, is well characterized (16). Type II IPP isomerase, found in Archaea and some of the Bacteria, was discovered much more recently (10), and only a few studies of the protein have been reported. Analysis of the genome of Synechocystis sp. strain PCC 6803 indicated that the cyanobacterium synthesizes isoprenoid compounds by the MEP route. Although it was reported elsewhere that the bacterium does not have detectable IPP isomerase activity under phototrophic growth conditions (6, 7), open reading frame (ORF) sll1556 encodes a protein that is substantially similar to other type II IPP isomerases. We now report that purified ORF sll1556 protein is an active type II IPP isomerase.

Evidence that Synechocystis sp. strain PCC 6803 ORF sll1556 encodes a functional type II IPP isomerase was obtained by complementation studies with Salmonella enterica serovar Typhimurium strain RMC29 (Table 1) (2). The chromosomal copy of idi (IPP isomerase) in RMC29 was disrupted with a chloramphenicol (CAM) marker, and dxs (deoxyxylulose synthase) was disrupted with a minioperon that includes the yeast genes required for biosynthesis of IPP from MVA and an ampicillin (AMP) marker. Thus, RMC29 is viable when supplemented with methylerythritol (ME) but does not grow on MVA unless idi activity is restored. RMC29 was transformed with plasmid pJMSB02278-8 bearing a copy of ORF sll1556 from Synechocystis sp. strain PCC 6803 and the expression plasmid without ORF sll1556. Both strains were resistant to AMP and expressed the plasmid-carried genes when induced with arabinose. As shown in Fig. 1, strains JMSB02278-8, JMSB0354, and RMC29 grew on LB broth-CAM-ME, demonstrating that they can utilize the MEP pathway to synthesize IPP and DMAPP without a functional IPP isomerase. Strains JMSB02278-8 and JMSB0354 contain plasmids conferring resistance to AMP and grew on LB-AMP-CAM-ME, while RMC29 did not. Only JMSB02278-8 grew on LB-AMP-CAM-MEV-arabinose by utilizing the IPP isomerase encoded by ORF sll1556 from Synechocystis sp. strain PCC 6803 to convert IPP synthesized from MVA to DMAPP.

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TABLE 1. Bacterial strains and plasmids

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FIG. 1. Complementation of an idi::MVA knockout in S. enterica serovar Typhimurium with ORF sll1556 from Synechocystis sp. strain 6803. Strains are RMC29 (dxs::MVA operon; idi::CAM), JMSB0354 (RMC29; Ampr), and JMSB02278-8 (RMC29; Ampr; ORF sll1556). AR, arabinose.

Nickel-nitrilotriacetic acid affinity chromatography of the supernatant from a cell extract of Escherichia coli strain JMSB0373a yielded a protein that gave a single band upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular mass of 50 kDa. This band was isolated, and an in-gel tryptic digestion was performed. Analysis of the matrix-assisted laser desorption ionization mass spectrometry of the tryptic peptides revealed 69% coverage of the amino acids from the expressed sequence of ORF sll1556 from Synechocystis sp. strain PCC 6803 (data not shown). The state of aggregation of the protein was examined by sedimentation equilibrium. The data were best described by a tetramer-octamer equilibrium, with K_d being 0.2 µM (Fig. 2). The protein exists in a homotetrameric state at the concentrations used for assays (29 nM). Isomerase activity was measured by the acid lability technique (2). The optimal pH and concentrations for Mg²⁺, flavin mononucleotide, and NADPH were determined, and under these conditions (37°C, 50 mM HEPES buffer [pH 7.0], 20 µM flavin mononucleotide, 10 mM NADPH, 20 mM MgCl₂, 50 µM dithiothreitol), K_m^IPP was 52 µM, k_cat^IPP was 0.23 s^–1, and k_cat/K_m was 4.4 x 10³ M^–1 s^–1.

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FIG. 2. Sedimentation equilibrium data for Synechocystis sp. strain 6803 type II IPP isomerase. The lower panel shows experimental data points for three different loading concentrations (, 2.8 µM; , 1.4 µM; , 0.7 µM) of the protein with the corresponding calculated fit (dotted line). The upper panels show the residuals for these fits; all are small and random, indicating a good fit corresponding to a Kd of 0.2 µM.

Isoprenoid metabolites play major roles during photosynthesis (8). Carotenoids are essential for the normal development of photosynthetic membranes, for dissipation of light energy, and for protecting plants from excess light (3-5, 15). Carotenoids quench singlet oxygen, and their absence is lethal in plants and oxygenic photosynthetic bacteria such as Synechocystis sp. strain PCC 6803. Chlorophyll is anchored to membranes by a hydrophobic phytyl chain. DMAPP is the essential allylic diphosphate primer for the biosynthesis of geranylgeranyl diphosphate required for carotenoids and phytol. In the MEP pathway DMAPP and IPP are synthesized simultaneously from hydroxydimethylallyl diphosphate to give a nonequilibrium (6:1) mixture favoring IPP (1). The isomerization of IPP and DMAPP results in a 1:2.5 ratio of IPP to DMAPP (14). In those organisms containing the MEP pathway and a type II IPP isomerase, such as cyanobacteria, the ratio of DMAPP relative to IPP can be increased in order to balance the pools of IPP and DMAPP during periods of high carotenoid biosynthesis where only three molecules of IPP are required for each DMAPP in the chain elongation reaction to produce geranylgeranyl diphosphate.

Prior to the discovery of type II IPP isomerase, Gannt and Ershov reported that the chromosome of Synechocystis sp. strain PCC 6803 did not appear to encode an IPP isomerase and that a cell extract from the bacterium did not have IPP isomerase activity (6). While this paper was under review, Poliquin and coworkers reported that the Synechocystis ORF sll1556 protein was inactive in a type II IPP isomerase assay, although the Streptomyces type II enzyme was active under the same conditions (12). This is in marked contrast to our findings for the ORF sll1556 protein, which has IPP isomerase activity comparable to that of other type II enzymes (Table 2). Given the similarities between the constructs and protocols used to obtain purified protein and the procedures used to assay for activity, we cannot explain the difference between our results and theirs.

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TABLE 2. Kinetic constants for type II IPP isomerasesa

 
We thank Cindy Chepanoske for providing matrix-assisted laser desorption ionization-time of flight mass spectra and Lisa Jones for assistance with the sedimentation equilibrium experiments.

This work was supported by Public Health Service grant GM 25521 from the Institute of General Medical Sciences.

 
* Corresponding author. Mailing address: 315 South 1400 East RM 2020, Department of Chemistry, University of Utah, Salt Lake City, UT 84112. Phone: (801) 581-6685. Fax: (801) 581-4391. E-mail: poulter{at}chem.utah.edu.

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Journal of Bacteriology, December 2004, p. 8156-8158, Vol. 186, No. 23
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.23.8156-8158.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Barkley, S. J. Articles by Poulter, C. D. Search for Related Content PubMed PubMed Citation Articles by Barkley, S. J. Articles by Poulter, C. D.