SUMMARY About 30 years have now passed since it was discovered that microbes synthesize RubisCO molecules that differ from the typical plant paradigm. RubisCOs of forms I, II, and III catalyze CO2 fixation reactions, albeit for potentially different physiological purposes, while the RubisCO-like protein (RLP) (form IV RubisCO) has evolved, thus far at least, to catalyze reactions that are important for sulfur metabolism. RubisCO is the major global CO2 fixation catalyst, and RLP is a somewhat related protein, exemplified by the fact that some of the latter proteins, along with RubisCO, catalyze similar enolization reactions as a part of their respective catalytic mechanisms. RLP in some organisms catalyzes a key reaction of a methionine salvage pathway, while in green sulfur bacteria, RLP plays a role in oxidative thiosulfate metabolism. In many organisms, the function of RLP is unknown. Indeed, there now appear to be at least six different clades of RLP molecules found in nature. Consideration of the many RubisCO (forms I, II, and III) and RLP (form IV) sequences in the database has subsequently led to a coherent picture of how these proteins may have evolved, with a form III RubisCO arising from the Methanomicrobia as the most likely ultimate source of all RubisCO and RLP lineages. In addition, structure-function analyses of RLP and RubisCO have provided information as to how the active sites of these proteins have evolved for their specific functions.

There are four forms of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) found in nature. Forms I, II, and III catalyse the carboxylation and oxygenation of ribulose 1,5-bisphosphate, while form IV, also called the Rubisco-like protein (RLP), does not catalyse either of these reactions. There appear to be six different clades of RLP. Although related to bona fide Rubisco proteins at the primary sequence and tertiary structure levels, RLP from two of these clades is known to perform other functions in the cell. Forms I, II, and III Rubisco, along with form IV (RLP), are thought to have evolved from a primordial archaeal Rubisco. Structure/function studies with both archaeal form III (methanogen) and form I (cyanobacterial) Rubisco have identified residues that appear to be specifically involved with interactions with molecular oxygen. A specific region of all form I, II, and III Rubisco was identified as being important for these interactions.

Ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RubisCO) catalyses the key reaction by which inorganic carbon may be assimilated into organic carbon. Phylogenetic analyses indicate that there are three classes of bona fide RubisCO proteins, forms I, II and III, which all catalyse the same reactions. In addition, there exists another form of RubisCO, form IV, which does not catalyse RuBP carboxylation or oxygenation. Form IV is actually a homologue of RubisCO and is called the RubisCO-like protein (RLP). Both RubisCO and RLP appear to have evolved from an ancestor protein in a methanogenic archaeon, and comprehensive analyses indicate that the different forms (I, II, III and IV ) contain various subgroups, with individual sequences derived from representatives of all three kingdoms of life. The diversity of RubisCO molecules, many of which function in distinct milieus, has provided convenient model systems to study the ways in which the active site of this protein has evolved to accommodate necessary molecular adaptations. Such studies have proven useful to help provide a framework for understanding the molecular basis for many important aspects of RubisCO catalysis, including the elucidation of factors or functional groups that impinge on RubisCO carbon dioxide/oxygen substrate discrimination.

Metagenomic studies recently uncovered form II/III RubisCO genes, originally thought to only occur in archaea, from uncultivated bacteria of the candidate phyla radiation (CPR). There are no isolated CPR bacteria and these organisms are predicted to have limited metabolic capacities. Here we expand the known diversity of RubisCO from CPR lineages. We report a form of RubisCO, distantly similar to the archaeal form III RubisCO, in some CPR bacteria from the Parcubacteria (OD1), WS6 and Microgenomates (OP11) phyla. In addition, we significantly expand the Peregrinibacteria (PER) II/III RubisCO diversity and report the first II/III RubisCO sequences from the Microgenomates and WS6 phyla. To provide a metabolic context for these RubisCOs, we reconstructed near-complete (>93%) PER genomes and the first closed genome for a WS6 bacterium, for which we propose the phylum name Dojkabacteria. Genomic and bioinformatic analyses suggest that the CPR RubisCOs function in a nucleoside pathway similar to that proposed in Archaea. Detection of form II/III RubisCO and nucleoside metabolism gene transcripts from a PER supports the operation of this pathway in situ. We demonstrate that the PER form II/III RubisCO is catalytically active, fixing CO2 to physiologically complement phototrophic growth in a bacterial photoautotrophic RubisCO deletion strain. We propose that the identification of these RubisCOs across a radiation of obligately fermentative, small-celled organisms hints at a widespread, simple metabolic platform in which ribose may be a prominent currency.

Ribulose-1,5-bisphosphate carboxylase͞oxygenase (Rubisco) catalyzes the rate-limiting step of photosynthetic CO 2 fixation and, thus, limits agricultural productivity. However, Rubisco enzymes from different species have different catalytic constants. If the structural basis for such differences were known, a rationale could be developed for genetically engineering an improved enzyme. Residues at the bottom of the large-subunit ␣͞␤-barrel active site of Rubisco from the green alga Chlamydomonas reinhardtii (methyl-Cys-256, Lys-258, and Ile-265) were previously changed through directed mutagenesis and chloroplast transformation to residues characteristic of land-plant Rubisco (Phe-256, Arg-258, and Val-265). The resultant enzyme has decreases in carboxylation efficiency and CO 2͞O2 specificity, despite the fact that land-plant Rubisco has greater specificity than the Chlamydomonas enzyme. Because the residues are close to a variable loop between ␤-strands A and B of the small subunit that can also affect catalysis, additional substitutions were created at this interface. When largesubunit Val-221 and Val-235 were changed to land-plant Cys-221 and Ile-235, they complemented the original substitutions and returned CO 2͞O2 specificity to the normal level. Further substitution with the shorter ␤A-␤B loop of the spinach small subunit caused a 12-17% increase in specificity. The enhanced CO 2͞O2 specificity of the mutant enzyme is lower than that of the spinach enzyme, but the carboxylation and oxygenation kinetic constants are nearly indistinguishable from those of spinach and substantially different from those of Chlamydomonas Rubisco. Thus, this interface between large and small subunits, far from the active site, contributes significantly to the differences in catalytic properties between algal and land-plant Rubisco enzymes.catalysis ͉ Chlamydomonas ͉ chloroplast ͉ photosynthesis ͉ ribulosebisphosphate carboxylase͞oxygenase C O 2 and O 2 compete at the active site of ribulose-1,5-bisphosphate (RuBP) carboxylase͞oxygenase [Ribulose-1,5-bisphosphate carboxylase͞oxygenase (Rubisco), Enzyme Commission 4.1.1.39] for either the carboxylation or oxygenation of RuBP (reviewed in refs. 1-3). Whereas carboxylation is responsible for the accumulation of carbon in the biosphere, oxygenation is a nonessential reaction that leads to the loss of fixed carbon via the photorespiratory pathway. The ratio of the catalytic efficiencies (V max ͞K m ) of carboxylation (V c ͞K c ) and oxygenation (V o ͞K o ) defines the CO 2 ͞O 2 -specificity kinetic constant ⍀ (4), which is determined by the differential stabilization of the carboxylation and oxygenation transition states for the rate-limiting partial reactions (5). However, net CO 2 fixation is determined by the difference between the velocities of carboxylation and oxygenation at the CO 2 and O 2 concentrations that occur in vivo (4, 6). Because of its pivotal role in catalyzing the rate-limiting step of photosynthesis, genetic engineering of Rubisco aimed at increasing net CO 2 fixa...