Purification and Characterization of Phosphoribulokinase from the Marine Chromophytic Alga Heterosigma carterae1

Abstract

In this study we characterized phosphoribulokinase (PRK, EC 2.7.1.19) from the eukaryotic marine chromophyte Heterosigma carterae. Serial column chromatography resulted in approximately 300-fold purification of the enzyme. A polypeptide of 53 kD was identified as PRK by sequencing the amino terminus of the protein. This protein represents one of the largest composite monomers identified to date for any PRK. The native holoenzyme demonstrated by flow performance liquid chromatography a molecular mass of 214 ؎ 12.6 kD, suggesting a tetrameric structure for this catalyst. Because H. carterae PRK activity was insensitive to NADH but was stimulated by dithiothreitol, it appears that the enzyme may require a thioredoxin/ferredoxin rather than a metabolite mode of regulation. Kinetic analysis of this enzyme demonstrated Michaelis constant values of ribulose-5-phosphate (226 M) and ATP (208 M), respectively. In summary, H. carterae PRK is unique with respect to holoenzyme structure and function, and thus may represent an alternative evolutionary pathway in Calvin-cycle kinase development.

Diatom Plastids Possess a Phosphoribulokinase with an Altered Regulation and No Oxidative Pentose Phosphate Pathway

“…The PRK purified by this procedure had a high specific activity (300 units mg 21 ), which is comparable to the enzyme purified from spinach (Gardemann et al, 1983; Table I). V max and the respective K m values for ATP and Ru5P were rather similar to the enzyme from spinach and the cyanobacterium Synechocystis PCC7942 but differed markedly from data published for the haptophyte alga H. carterae (Hariharan et al, 1998).…”

“…By gel filtration experiments, we found that the PRKs from different diatoms (O. sinensis, Coscinodiscus granii, and P. tricornutum) are homodimers with an apparent molecular mass of 44 kD/subunits on SDS gels (data not shown). This is identical to PRK enzymes from land plants but differs from previously published data for the PRK of the haptophyte Heterosigma carterae (Hariharan et al, 1998).…”

“…The putative N terminus of the mature O. sinensis PRK protein (position 43) was inferred by comparison of the complete amino acid sequence of the PRK with (1) other mature PRK sequences (Milanez and Mural, 1988;Hariharan et al, 1998) and (2) known cleavage motifs in diatom plastid preproteins (Kroth, 2002). The PRK was expressed in Escherichia coli as soluble protein but only in low amounts.…”

Chloroplast genome sequencing analysis of Heterosigma akashiwo CCMP452 (West Atlantic) and NIES293 (West Pacific) strains

“…As a coastal-dwelling organism, H. akashiwo also contributes significantly to primary productivity within these critically important ecosystems [27]. Significant research on its morphology [28], physiology [29-31], molecular biology [32-34], toxicology [35,36], and biochemistry [37-39] define H. akashiwo as one of the most broadly studied non-diatomaceous stramenopiles. Much of this attention has been focused on events associated with chloroplast biology.…”

Phosphoribulokinase: Current Perspectives on the Structure/Function Basis for Regulation and Catalysis

“…The two-fold symmetry that relates the top to the bottom layer creates the impression of a fourbladed pinwheel, with each pinwheel blade (e.g., lightly shaded subunits at right side of figure) corresponding to the dimer believed to represent the eukaryotic form of PRK. (Hariharan et al, 1998) suggests that this dithiothreitol (DTT)-stimulated enzyme is a tetramer of 53 kDa subunits. If such an assignment is confirmed, this protein would represent a new PRK quaternary structure.…”