Discovery of a RuBisCO-like Protein that Functions as an Oxygenase in the Novel D-Hamamelose Pathway

Abstract

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is the key catalyst of CO2 fixation and the most abundant protein found in nature. There are four forms of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). RuBisCO forms I, II, and III catalyse CO2 fixation reactions, whereas form IV, also called the RuBisCO-like protein (RLP), is known to have no carboxylase or oxygenase activities. RLPs are further divided into seven phylogenetically distinct subgroups: IV-Nonphoto (found in nonphototrophic bacteria), IV-Photo (found in phototrophic bacteria), IV-AMC (acid mine consortia), IV-GOS (global ocean sequencing sequencing program), IV-DeepYkr, IV-Aful (Archaeoglobus fulgidus), and IV-YkrW. Although numerous RLP genes have been found in bacterial and archaeal genomes, their roles and functions still remain largely unknown. To date, only two RLPs from Bacillus subtilis BSU13590 (IV-YkrW) and Rhodospirillum rubrum Rru_A1998 (IV-DeepYkr) have been shown to catalyse defined reactions in the methionine salvage pathway and S-adenosyl methionine metabolism, respectively. Here, we demonstrate that an RLP acts as an oxygenase in the novel D-hamamelose (2-C-hydroxymethyl-D-ribose) metabolic pathway by converting 3-keto-D-ribitol-1,5-bisphosphate (KRBP), an isomer of ribulose-1,5-bisphosphate (RuBP), to two phosphorylated hydroxy-acids, 3-D-phosphoglycerate and phosphoglycolate. Through bioinformatic analysis of the microbial genomes, we found that the gene cluster in Ochrobactrum anthropi ATCC 49188 contains a putative operon which possesses the RLP gene belonging to the IV-Nonphoto subgroup and a putative oxidoreductase gene (COG0673), the cluster of orthologous groups (COG) number of which is identical to that of sugar-oxidising enzymes, including inositol dehydrogenase and D-xylose dehydrogenase. In addition, the ATP-binding cassette (ABC) transporters located next to the oxidoreductase are homologous to those in the erythritol operon. Nine genes encoding four ABC transporter proteins (Oant_3061-3064) and five metabolic enzymes (Oant_3059, 3060, 3065-3067) constitute the gene cluster of O. anthropi. We hypothesized that the RLP in this gene cluster may be involved in sugar metabolism and that the RLPs belonging to the IV-Nonphoto subgroup may show catalytic activities similar to RuBisCO because the IV-Nonphoto clade is closest to RuBisCO forms I, II, and III. By applying the bioinformatic and genomic context analyses that we assigned the putative functions of Oant_3059, Oant_3060, Oant_3065, Oant_3066, and Oant_3067 genes as sugar lactonase, sugar dehydrogenase, sugar-acid phosphate dehydrogenase, sugar-acid kinase, and a RuBisCO-like enzyme that acts on ketose phosphate, respectively. The predicted pathway was thought to belong to the semiphosphorylative pathway, in which the first two steps are nonphosphrylative and the remaining three steps are phosphorylative. To experimentally demonstrate the predicted functions of these enzymes, aforementioned five genes were heterologously expressed. The putative operon with this RLP gene has been shown to be involved in the breakdown of D-hamamelose. D-Hamamelose is known to be present in most higher plants. The D-hamamelose pathway is comprised of five previously unknown enzymes: D-hamamelose dehydrogenase (Oant_3060, HamH), D-hamamelono-lactonase (Oant_3059, HamI), D-hamamelonate kinase (Oant_3066, HamB), D-hamamelonate-2,5-bisphosphate (decarboxylating) dehydrogenase (Oant_3065, HamC), and the RLP 3-keto-D-ribitol-1,5-bisphosphate (KRBP) oxygenase (Oant_3067, HamA), which converts KRBP to 3-D-phosphoglycerate and phosphoglycolate. We demonstrated, for the first time, the existence of a RuBisCO-like oxygenase (IV-Nonphoto RLP) in O. anthropi. Unlike RuBisCO, which has both carboxylase and oxygenase activities, the RLP from O. anthropi (Oant_3067) served only as an oxygenase with no detectable carboxylase activity and converted KRBP to 3-D-phosphoglycerate and phosphoglycolate. HamA represents the first RLP catalyzing the O2-dependent oxidative C–C bond cleavage reaction, and our findings may provide insights into its applications in oxidative C–C bond cleavage of organic molecules. One major advantage of RLP-catalyzed oxygenation is that the reaction occurs in the absence of organic cofactors such as flavins or pterins. In addition, our findings may provide insights into applications in the fields of biotechnology and bioengineering. Currently, improving the carboxylation yield of RuBisCO is a challenge because of its lower catalytic efficiency. More than one third of RuBP molecules are oxygenated rather than carboxylated. Thus, a better understanding of the reaction mechanism of RLP oxygenases may help to establish new methods for reducing the oxygenation activity of RuBisCO and thereby affect the production yields of crop plants.