D-Ribulose-5-phosphate 3-epimerase: cloning and heterologous expression of the spinach gene, and purification and characterization of the recombinant enzyme

Abstract

We have achieved, to our knowledge, the first high-level heterologous expression of the gene encoding D-ribulose-5-phosphate 3-epimerase from any source, thereby permitting isolation and characterization of the epimerase as found in photosynthetic organisms. The extremely labile recombinant spinach (Spinacia oleracea L.) enzyme was stabilized by DL-alpha-glycerophosphate or ethanol and destabilized by D-ribulose-5-phosphate or 2-mercaptoethanol. Despite this lability, the unprecedentedly high specific activity of the purified material indicates that the structural integrity of the enzyme is maintained throughout isolation. Ethylenediaminetetraacetate and divalent metal cations did not affect epimerase activity, thereby excluding a requirement for the latter in catalysis. As deduced from the sequence of the cloned spinach gene and the electrophoretic mobility under denaturing conditions of the purified recombinant enzyme, its 25-kD subunit size was about the same as that of the corresponding epimerases of yeast and mammals. However, in contrast to these other species, the recombinant spinach enzyme was octameric rather than dimeric, as assessed by gel filtration and polyacrylamide gel electrophoresis under nondenaturing conditions. Western-blot analyses with antibodies to the purified recombinant enzyme confirmed that the epimerase extracted from spinach leaves is also octameric.

Figure 1

Sequence of expression cassettes for spinach recombinant Ru5P epimerase. A, Transit protein expression cassette (accession no. AF070942). The tac promoter, operator, and ribosome-binding site (rbs) are shown in lowercase; the coding sequence is in uppercase. B, Mature epimerase expression cassette (accession no. AF070943).

Figure 2

Resolution of two peaks of epimerase by anion-exchange chromatography on Mono-Q. See text for additional details.

Figure 3

Progression of purification of spinach recombinant epimerase as assessed by SDS-PAGE. A, Gel stained with Coomassie blue includes: lane 1, molecular mass markers (bands from top to bottom: phosphorylase b, 94 kD; albumin, 67 kD; ovalbumin, 43 kD; carbonic anhydrase, 30 kD; trypsin inhibitor, 20.1 kD; lactalbumin, 14.4 kD); lane 2, centrifuged extract (3 μg); lane 3, pool from DE52 (2 μg); and lane 4, pool from hydroxyapatite (0.5 μg). B, Gel stained with silver for increased sensitivity includes: lane 1, peak I from Mono-Q (20 ng); and lane 2, peak II from Mono-Q (20 ng).

Figure 4

Purity and molecular mass of recombinant epimerase as assessed by PAGE under nondenaturing conditions. The Coomassie blue-stained gel includes: lane 1, molecular mass markers (bands from top to bottom: thyroglobulin, 669 kD; ferritin, 440 kD; catalase 232 kD; lactate dehydrogenase, 140 kD; albumin, 67 kD); lane 2, peak I from Mono-Q (1 μg); and lane 3, peak II from Mono-Q (1 μg).

Figure 5

IEF of recombinant epimerase under denaturing (A) and nondenaturing (B) conditions. Lanes 1 and 2 in both Coomassie blue-stained gels represent peaks I and II, respectively, from Mono-Q chromatography (Fig. 2). Sample loads were 0.6 μg in A and 1.0 μg in B. pI markers in B include (from top to bottom) human carbonic anhydrase B (pI 6.55), bovine carbonic anhydrase B (pI 5.85), β-lactoglobulin A (pI 5.2), and soybean trypsin inhibitor (pI 4.55).

Figure 6

Inherent instability of recombinant epimerase and effects of additives. All incubations were at 2°C in a pH 8.0 buffer of 50 mm Bicine. With the exception the one data set depicting centrifuged extract (250 μg total protein mL⁻¹) without any additives (○), all others depict purified enzyme (50 μg mL⁻¹): no additives (▪), 10 mm G3P introduced at 0 time or after 2 h (□), 20% (v/v) ethanol (▵), 1 mm Ru5P (•), and 10 mm 2-mercaptoethanol (▴). See text for additional details.

Figure 7

Western-blot analyses of purified recombinant epimerase, E. coli extracts, and spinach leaf extract subjected to SDS-PAGE (A), nondenaturing PAGE (B), or nondenaturing IEF (C). In A, samples are as follows: lane 1, peak I from Mono-Q (0.12 μg); lane 2, centrifuged extract from transformed E. coli (2.5 μg); lane 3, centrifuged extract from spinach (20 μg); and lane 4, prestained molecular mass markers (bands from top to bottom: myosin, 250 kD; phosphorylase b, 148 kD; glutamic dehydrogenase, 60 kD; carbonic anhydrase, 42 kD; myoglobin, 22 kD; lysozyme, 17 kD; aprotinin, 6 kD). In B, samples are as follows: lane 1, peak I from Mono-Q (15 ng); lane 2, peak II from Mono-Q (15 ng); lane 3, centrifuged extract from nontransformed E. coli (0.5 μg) (note the absence of cross-reactive material); lane 4, centrifuged extract from transformed E. coli (0.5 μg); and lane 5, centrifuged extract from spinach (15 μg). In C, samples are as follows: lane 1, peak I from Mono-Q (0.5 μg); and lane 2, peak II from Mono-Q (0.5 μg).