Abstract
The sequences classified as genes for various ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBisCO)-like proteins (RLPs) are widely distributed among bacteria, archaea, and eukaryota. In the phylogenic tree constructed with these sequences, RuBisCOs and RLPs are grouped into four separate clades, forms I-IV. In RuBisCO enzymes encoded by form I, II, and III sequences, 19 conserved amino acid residues are essential for CO(2) fixation; however, 1-11 of these 19 residues are substituted with other amino acids in form IV RLPs. Among form IV RLPs, the only enzymatic activity detected to date is a 2,3-diketo-5-methylthiopentyl 1-phosphate (DK-MTP-1-P) enolase reaction catalyzed by Bacillus subtilis, Microcystis aeruginosa, and Geobacillus kaustophilus form IV RLPs. RLPs from Rhodospirillum rubrum, Rhodopseudomonas palustris, Chlorobium tepidum, and Bordetella bronchiseptica were inactive in the enolase reaction. DK-MTP-1-P enolase activity of B. subtilis RLP required Mg(2+) for catalysis and, like RuBisCO, was stimulated by CO(2). Four residues that are essential for the enolization reaction of RuBisCO, Lys(175), Lys(201), Asp(203), and Glu(204), were conserved in RLPs and were essential for DK-MTP-1-P enolase catalysis. Lys(123), the residue conserved in DK-MTP-1-P enolases, was also essential for B. subtilis RLP enolase activity. Similarities between the active site structures of RuBisCO and B. subtilis RLP were examined by analyzing the effects of structural analogs of RuBP on DK-MTP-1-P enolase activity. A transition state analog for the RuBP carboxylation of RuBisCO was a competitive inhibitor in the DK-MTP-1-P enolase reaction with a K(i) value of 103 mum. RuBP and d-phosphoglyceric acid, the substrate and product, respectively, of RuBisCO, were weaker competitive inhibitors. These results suggest that the amino acid residues utilized in the B. subtilis RLP enolase reaction are the same as those utilized in the RuBisCO RuBP enolization reaction.
Highlights
In nature, there are wide variations in the properties and primary sequences of RuBisCO among different photosynthetic organisms (9 –12)
Distribution of DK-MTP-1-P Enolase Activity among RuBisCO-like protein (RLP)— some bacteria, archaea, and eukaryota have been reported to possess RLP genes, the only known function of these RLPs is enolization of DK-MTP-1-P to 2-hydroxy-3-keto5-methylthiopentenyl 1-phosphate in RLPs from B. subtilis, G. kaustophilus, and M. aeruginosa classified in group ␣1 [20, 24, 25]
There is no information regarding the distribution of this function among other RLPs in other groups or regarding functional analysis of the amino acid residues that may be involved in the DK-MTP-1-P enolization reaction
Summary
RuBisCO-essential residues that are conserved in RLPs (Fig. 1B). Form IV RLPs are further subdivided into four groups; ␣1, ␣2, , and ␥ [21]. As is the case in RuBisCO, the enolate intermediate is stabilized by Mg2ϩ and several amino acid residues: Lys175, Asp203, Glu204, His294, and the carbamylated Lys201 The results of these studies suggest that the DK-MTP-1-P enolase is structurally and functionally related to photosynthetic RuBisCO. It has been reported that when lysine at 201 is substituted with an alanine in the G. kaustophilus RLP, the enzyme is still capable of catalyzing enolization of DK-MTP-1-P [25] This result raises a question about the above hypothesis on the close evolutionary relationship between the RLP and RuBisCO, because a carbamylated lysine residue would be required at this position to form the Mg2ϩ-chelating triad linkage together with Asp203 and Glu204 and to stabilize the reaction intermediate in the RuBP enolization reaction of RuBisCO. The fact that the transition state analog of RuBisCO interacts with the active site of Bacillus RLP strongly supports their evolutionary proximity
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