Microbial pathway for anaerobic 5'-methylthioadenosine metabolism coupled to ethylene formation.

Numerous cellular processes involving S-adenosyl-l-methionine result in the formation of the toxic by-product, 5'-methylthioadenosine (MTA). To prevent inhibitory MTA accumulation and retain biologically available sulfur, most organisms possess the "universal" methionine salvage pathway (MSP). However, the universal MSP is inherently aerobic due to a requirement of molecular oxygen for one of the key enzymes. Here, we report the presence of an exclusively anaerobic MSP that couples MTA metabolism to ethylene formation in the phototrophic bacteria Rhodospirillum rubrum and Rhodopseudomonas palustris In vivo metabolite analysis of gene deletion strains demonstrated that this anaerobic MSP functions via sequential action of MTA phosphorylase (MtnP), 5-(methylthio)ribose-1-phosphate isomerase (MtnA), and an annotated class II aldolase-like protein (Ald2) to form 2-(methylthio)acetaldehyde as an intermediate. 2-(Methylthio)acetaldehyde is reduced to 2-(methylthio)ethanol, which is further metabolized as a usable organic sulfur source, generating stoichiometric amounts of ethylene in the process. Ethylene induction experiments using 2-(methylthio)ethanol versus sulfate as sulfur sources further indicate anaerobic ethylene production from 2-(methylthio)ethanol requires protein synthesis and that this process is regulated. Finally, phylogenetic analysis reveals that the genes corresponding to these enzymes, and presumably the pathway, are widespread among anaerobic and facultatively anaerobic bacteria from soil and freshwater environments. These results not only establish the existence of a functional, exclusively anaerobic MSP, but they also suggest a possible route by which ethylene is produced by microbes in anoxic environments.

Structural Perturbations of Rhodopseudomonas palustris Form II RuBisCO Mutant Enzymes That Affect CO2 Fixation.

The enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and its central role in capturing atmospheric CO2 via the Calvin-Benson-Bassham (CBB) cycle have been well-studied. Previously, a form II RuBisCO from Rhodopseudomonas palustris, a facultative anaerobic bacterium, was shown to assemble into a hexameric holoenzyme. Unlike previous studies with form II RuBisCO, the R. palustris enzyme could be crystallized in the presence of the transition state analogue 2-carboxyarabinitol 1,5-bisphosphate (CABP), greatly facilitating the structure-function studies reported here. Structural analysis of mutant enzymes with substitutions in form II-specific residues (Ile165 and Met331) and other conserved and semiconserved residues near the enzyme's active site identified subtle structural interactions that may account for functional differences between divergent RuBisCO enzymes. In addition, using a distantly related aerobic bacterial host, further selection of a suppressor mutant enzyme that overcomes negative enzymatic functions was accomplished. Structure-function analyses with negative and suppressor mutant RuBisCOs highlighted the importance of interactions involving different parts of the enzyme's quaternary structure that influenced partial reactions that constitute RuBisCO's carboxylation mechanism. In particular, structural perturbations in an intersubunit interface appear to affect CO2 addition but not the previous step in the enzymatic mechanism, i.e., the enolization of substrate ribulose 1,5-bisphosphate (RuBP). This was further substantiated by the ability of a subset of carboxylation negative mutants to support a previously described sulfur-salvage function, one that appears to rely solely on the enzyme's ability to catalyze the enolization of a substrate analogous to RuBP.

Isotope discrimination by form IC RubisCO from Ralstonia eutropha and Rhodobacter sphaeroides, metabolically versatile members of 'Proteobacteria' from aquatic and soil habitats.

RubisCO, the CO2 fixing enzyme of the Calvin-Benson-Bassham (CBB) cycle, is responsible for the majority of carbon fixation on Earth. RubisCO fixes 12 CO2 faster than 13 CO2 resulting in 13 C-depleted biomass, enabling the use of δ13 C values to trace CBB activity in contemporary and ancient environments. Enzymatic fractionation is expressed as an ε value, and is routinely used in modelling, for example, the global carbon cycle and climate change, and for interpreting trophic interactions. Although values for spinach RubisCO (ε = ~29‰) have routinely been used in such efforts, there are five different forms of RubisCO utilized by diverse photolithoautotrophs and chemolithoautotrophs and ε values, now known for four forms (IA, B, D and II), vary substantially with ε = 11‰ to 27‰. Given the importance of ε values in δ13 C evaluation, we measured enzymatic fractionation of the fifth form, form IC RubisCO, which is found widely in aquatic and terrestrial environments. Values were determined for two model organisms, the 'Proteobacteria' Ralstonia eutropha (ε = 19.0‰) and Rhodobacter sphaeroides (ε = 22.4‰). It is apparent from these measurements that all RubisCO forms measured to date discriminate less than commonly assumed based on spinach, and that enzyme ε values must be considered when interpreting and modelling variability of δ13 C values in nature.

The Arnon-Buchanan cycle: a retrospective, 1966-2016.

For the first decade following its description in 1954, the Calvin-Benson cycle was considered the sole pathway of autotrophic CO2 assimilation. In the early 1960s, experiments with fermentative bacteria uncovered reactions that challenged this concept. Ferredoxin was found to donate electrons directly for the reductive fixation of CO2 into alpha-keto acids via reactions considered irreversible. Thus, pyruvate and alpha-ketoglutarate could be synthesized from CO2, reduced ferredoxin and acetyl-CoA or succinyl-CoA, respectively. This work opened the door to the discovery that reduced ferredoxin could drive the Krebs citric acid cycle in reverse, converting the pathway from its historical role in carbohydrate breakdown to one fixing CO2. Originally uncovered in photosynthetic green sulfur bacteria, the Arnon-Buchanan cycle has since been divorced from light and shown to function in a variety of anaerobic chemoautotrophs. In this retrospective, colleagues who worked on the cycle at its inception in 1966 and those presently working in the field trace its development from a controversial reception to its present-day inclusion in textbooks. This pathway is now well established in major groups of chemoautotrophic bacteria, instead of the Calvin-Benson cycle, and is increasingly referred to as the Arnon-Buchanan cycle. In this retrospective, separate sections have been written by the authors indicated. Bob Buchanan wrote the abstract and the concluding comments.

In Vivo Studies in Rhodospirillum rubrum Indicate That Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) Catalyzes Two Obligatorily Required and Physiologically Significant Reactions for Distinct Carbon and Sulfur Metabolic Pathways.

All organisms possess fundamental metabolic pathways to ensure that needed carbon and sulfur compounds are provided to the cell in the proper chemical form and oxidation state. For most organisms capable of using CO2 as sole source of carbon, ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco) catalyzes primary carbon dioxide assimilation. In addition, sulfur salvage pathways are necessary to ensure that key sulfur-containing compounds are both available and, where necessary, detoxified in the cell. Using knock-out mutations and metabolomics in the bacterium Rhodospirillum rubrum, we show here that Rubisco concurrently catalyzes key and essential reactions for seemingly unrelated but physiologically essential central carbon and sulfur salvage metabolic pathways of the cell. In this study, complementation and mutagenesis studies indicated that representatives of all known extant functional Rubisco forms found in nature are capable of simultaneously catalyzing reactions required for both CO2-dependent growth as well as growth using 5-methylthioadenosine as sole sulfur source under anaerobic photosynthetic conditions. Moreover, specific inactivation of the CO2 fixation reaction did not affect the ability of Rubisco to support anaerobic 5-methylthioadenosine metabolism, suggesting that the active site of Rubisco has evolved to ensure that this enzyme maintains both key functions. Thus, despite the coevolution of both functions, the active site of this protein may be differentially modified to affect only one of its key functions.

Functional metagenomic selection of ribulose 1, 5-bisphosphate carboxylase/oxygenase from uncultivated bacteria.

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) is a critical yet severely inefficient enzyme that catalyses the fixation of virtually all of the carbon found on Earth. Here, we report a functional metagenomic selection that recovers physiologically active RubisCO molecules directly from uncultivated and largely unknown members of natural microbial communities. Selection is based on CO2 -dependent growth in a host strain capable of expressing environmental deoxyribonucleic acid (DNA), precluding the need for pure cultures or screening of recombinant clones for enzymatic activity. Seventeen functional RubisCO-encoded sequences were selected using DNA extracted from soil and river autotrophic enrichments, a photosynthetic biofilm and a subsurface groundwater aquifer. Notably, three related form II RubisCOs were recovered which share high sequence similarity with metagenomic scaffolds from uncultivated members of the Gallionellaceae family. One of the Gallionellaceae RubisCOs was purified and shown to possess CO2 /O2 specificity typical of form II enzymes. X-ray crystallography determined that this enzyme is a hexamer, only the second form II multimer ever solved and the first RubisCO structure obtained from an uncultivated bacterium. Functional metagenomic selection leverages natural biological diversity and billions of years of evolution inherent in environmental communities, providing a new window into the discovery of CO2 -fixing enzymes not previously characterized.

CbbR, the Master Regulator for Microbial Carbon Dioxide Fixation.

Biological carbon dioxide fixation is an essential and crucial process catalyzed by both prokaryotic and eukaryotic organisms to allow ubiquitous atmospheric CO2 to be reduced to usable forms of organic carbon. This process, especially the Calvin-Bassham-Benson (CBB) pathway of CO2 fixation, provides the bulk of organic carbon found on earth. The enzyme ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RubisCO) performs the key and rate-limiting step whereby CO2 is reduced and incorporated into a precursor organic metabolite. This is a highly regulated process in diverse organisms, with the expression of genes that comprise the CBB pathway (the cbb genes), including RubisCO, specifically controlled by the master transcriptional regulator protein CbbR. Many organisms have two or more cbb operons that either are regulated by a single CbbR or employ a specific CbbR for each cbb operon. CbbR family members are versatile and accommodate and bind many different effector metabolites that influence CbbR's ability to control cbb transcription. Moreover, two members of the CbbR family are further posttranslationally modified via interactions with other transcriptional regulator proteins from two-component regulatory systems, thus augmenting CbbR-dependent control and optimizing expression of specific cbb operons. In addition to interactions with small effector metabolites and other regulator proteins, CbbR proteins may be selected that are constitutively active and, in some instances, elevate the level of cbb expression relative to wild-type CbbR. Optimizing CbbR-dependent control is an important consideration for potentially using microbes to convert CO2 to useful bioproducts.

Structure-function studies with the unique hexameric form II ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Rhodopseudomonas palustris.

The first x-ray crystal structure has been solved for an activated transition-state analog-bound form II ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). This enzyme, from Rhodopseudomonas palustris, assembles as a unique hexamer with three pairs of catalytic large subunit homodimers around a central 3-fold symmetry axis. This oligomer arrangement is unique among all known Rubisco structures, including the form II homolog from Rhodospirillum rubrum. The presence of a transition-state analog in the active site locked the activated enzyme in a "closed" conformation and revealed the positions of critical active site residues during catalysis. Functional roles of two form II-specific residues (Ile(165) and Met(331)) near the active site were examined via site-directed mutagenesis. Substitutions at these residues affect function but not the ability of the enzyme to assemble. Random mutagenesis and suppressor selection in a Rubisco deletion strain of Rhodobacter capsulatus identified a residue in the amino terminus of one subunit (Ala(47)) that compensated for a negative change near the active site of a neighboring subunit. In addition, substitution of the native carboxyl-terminal sequence with the last few dissimilar residues from the related R. rubrum homolog increased the enzyme's kcat for carboxylation. However, replacement of a longer carboxyl-terminal sequence with termini from either a form III or a form I enzyme, which varied both in length and sequence, resulted in complete loss of function. From these studies, it is evident that a number of subtle interactions near the active site and the carboxyl terminus account for functional differences between the different forms of Rubiscos found in nature.

Amino acid substitutions in the transcriptional regulator CbbR lead to constitutively active CbbR proteins that elevate expression of the cbb CO2 fixation operons in Ralstonia eutropha (Cupriavidus necator) and identify regions of CbbR necessary for gene activation.

CbbR is a LysR-type transcriptional regulator that activates expression of the operons containing (cbb) genes that encode the CO2 fixation pathway enzymes in Ralstonia eutropha (Cupriavidus necator) under autotrophic growth conditions. The cbb operons are stringently downregulated during chemoheterotrophic growth on organic acids such as malate. CbbR constitutive proteins (CbbR*s), typically with single amino acid substitutions, were selected and isolated that activate expression of the cbb operons under chemoheterotrophic growth conditions. A large set of CbbR*s from all major domains of the CbbR molecule were identified, except for the DNA-binding domain. The level of gene expression conferred for many of these CbbR*s under autotrophic growth was greater than that conferred by wild-type CbbR. Several of these CbbR*s increase transcription two- to threefold more than wild-type CbbR. One particular CbbR*, a truncated protein, was useful in identifying the regions of CbbR that are necessary for transcriptional activation and, by logical extension, necessary for interaction with RNA polymerase. The reductive assimilation of carbon via CO2 fixation is an important step in the cost-effective production of useful biological compounds. Enhancing CO2 fixation in Ralstonia eutropha through greater transcriptional activation of the cbb operons could prove advantageous, and the use of CbbR*s is one way to enhance product formation.

Phosphoribulokinase mediates nitrogenase-induced carbon dioxide fixation gene repression in Rhodobacter sphaeroides.

In many organisms there is a balance between carbon and nitrogen metabolism. These observations extend to the nitrogen-fixing, nonsulfur purple bacteria, which have the classic family of P(II) regulators that coordinate signals of carbon and nitrogen status to regulate nitrogen metabolism. Curiously, these organisms also possess a reverse mechanism to regulate carbon metabolism based on cellular nitrogen status. In this work, studies in Rhodobacter sphaeroides firmly established that the activity of the enzyme that catalyses nitrogen fixation, nitrogenase, induces a signal that leads to repression of genes encoding enzymes of the Calvin-Benson-Bassham (CBB) CO2 fixation pathway. Additionally, genetic and metabolomic experiments revealed that NADH-activated phosphoribulokinase is an intermediate in the signalling pathway. Thus, nitrogenase activity appears to be linked to cbb gene repression through phosphoribulokinase.

Amino acid residues of RegA important for interactions with the CbbR-DNA complex of Rhodobacter sphaeroides.

CbbR and RegA (PrrA) are transcriptional regulators of the Calvin-Benson-Bassham (CBB) CO2 fixation pathway (cbbI and cbbII) operons of Rhodobacter sphaeroides. The CbbR and RegA proteins interact, but CbbR must be bound to the promoter DNA in order for RegA-CbbR protein-protein interactions to occur. RegA greatly enhances the ability of CbbR to bind the cbbI promoter or greatly enhances the stability of the CbbR/promoter complex. The N-terminal receiver domain and the DNA binding domain of RegA were shown to interact with CbbR. Residues in α-helix 7 and α-helix 8 of the DNA binding domain (helix-turn-helix) of RegA directly interacted with CbbR, with α-helix 7 positioned immediately above the DNA and α-helix 8 located in the major groove of the DNA. A CbbR protein containing only the DNA binding motif and the linker helix was capable of binding to RegA. In contrast, a truncated CbbR containing only the linker helix and recognition domains I and II (required for effector binding) was not able to interact with RegA. The accumulated results strongly suggest that the DNA binding domains of both proteins interact to facilitate optimal transcriptional control over the cbb operons. In vivo analysis, using constitutively active mutant CbbR proteins, further indicated that CbbR must interact with phosphorylated RegA in order to accomplish transcriptional activation.

Altered residues in key proteins influence the expression and activity of the nitrogenase complex in an adaptive CO2 fixation-deficient mutant strain of Rhodobacter sphaeroides.

Previously, the RubisCO-compromised spontaneous adaptive Rhodobacter sphaeroides mutant, strain 16PHC, was shown to derepress the expression of genes that encode the nitrogenase complex under normal repressive conditions. As a result of this adaptation, the active nitrogenase complex restored redox balance, thus allowing strain 16PHC to grow under photoheterotrophic conditions in the absence of an exogenous electron acceptor. A combination of whole genome pyrosequencing and whole genome microarray analyses was employed to identify possible loci responsible for the observed phenotype. Mutations were found in two genes, glnA and nifA, whose products are involved in the regulatory cascade that controls nitrogenase complex gene expression. In addition, a nucleotide reversion within the nifK gene, which encodes a subunit of the nitrogenase complex, was also identified. Subsequent genetic, physiological and biochemical studies revealed alterations that led to derepression of the synthesis of an active nitrogenase complex in strain 16PHC.

A RubisCO-like protein links SAM metabolism with isoprenoid biosynthesis.

Functional assignment of uncharacterized proteins is a challenge in the era of large-scale genome sequencing. Here, we combine in extracto NMR, proteomics and transcriptomics with a newly developed (knock-out) metabolomics platform to determine a potential physiological role for a ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO)-like protein from Rhodospirillum rubrum. Our studies unraveled an unexpected link in bacterial central carbon metabolism between S-adenosylmethionine-dependent polyamine metabolism and isoprenoid biosynthesis and also provide an alternative approach to assign enzyme function at the organismic level.

Escherichia coli possessing the dihydroxyacetone phosphate shunt utilize 5'-deoxynucleosides for growth.

All organisms utilize S-adenosyl-l-methionine (SAM) as a key co-substrate for the methylation of biological molecules, the synthesis of polyamines, and radical SAM reactions. When these processes occur, 5'-deoxy-nucleosides are formed as byproducts such as S-adenosyl-l-homocysteine, 5'-methylthioadenosine (MTA), and 5'-deoxyadenosine (5dAdo). A prevalent pathway found in bacteria for the metabolism of MTA and 5dAdo is the dihydroxyacetone phosphate (DHAP) shunt, which converts these compounds into dihydroxyacetone phosphate and 2-methylthioacetaldehyde or acetaldehyde, respectively. Previous work in other organisms has shown that the DHAP shunt can enable methionine synthesis from MTA or serve as an MTA and 5dAdo detoxification pathway. Rather, the DHAP shunt in Escherichia coli ATCC 25922, when introduced into E. coli K-12, enables the use of 5dAdo and MTA as a carbon source for growth. When MTA is the substrate, the sulfur component is not significantly recycled back to methionine but rather accumulates as 2-methylthioethanol, which is slowly oxidized non-enzymatically under aerobic conditions. The DHAP shunt in ATCC 25922 is active under oxic and anoxic conditions. Growth using 5-deoxy-d-ribose was observed during aerobic respiration and anaerobic respiration with Trimethylamine N-oxide (TMAO), but not during fermentation or respiration with nitrate. This suggests the DHAP shunt may only be relevant for extraintestinal pathogenic E. coli lineages with the DHAP shunt that inhabit oxic or TMAO-rich extraintestinal environments. This reveals a heretofore overlooked role of the DHAP shunt in carbon and energy metabolism from ubiquitous SAM utilization byproducts and suggests a similar role may occur in other pathogenic and non-pathogenic bacteria with the DHAP shunt. IMPORTANCE: The acquisition and utilization of organic compounds that serve as growth substrates are essential for Escherichia coli to grow and multiply. Ubiquitous enzymatic reactions involving S-adenosyl-l-methionine as a co-substrate by all organisms result in the formation of the 5'-deoxy-nucleoside byproducts, 5'-methylthioadenosine and 5'-deoxyadenosine. All E. coli possess a conserved nucleosidase that cleaves these 5'-deoxy-nucleosides into 5-deoxy-pentose sugars for adenine salvage. The DHAP shunt pathway is found in some extraintestinal pathogenic E. coli, but its function in E. coli possessing it has remained unknown. This study reveals that the DHAP shunt enables the utilization of 5'-deoxy-nucleosides and 5-deoxy-pentose sugars as growth substrates in E. coli strains with the pathway during aerobic respiration and anaerobic respiration with TMAO, but not fermentative growth. This provides an insight into the diversity of sugar compounds accessible by E. coli with the DHAP shunt and suggests that the DHAP shunt is primarily relevant in oxic or TMAO-rich extraintestinal environments.

Regulatory twist and synergistic role of metabolic coinducer- and response regulator-mediated CbbR-cbbI interactions in Rhodopseudomonas palustris CGA010.

Rhodopseudomonas palustris assimilates CO2 by the Calvin-Benson-Bassham (CBB) reductive pentose phosphate pathway. Most genes required for a functional CBB pathway are clustered into the cbbI and cbbII operons, with the cbbI operon subject to control by a LysR transcriptional activator, CbbR, encoded by cbbR, which is divergently transcribed from the cbbLS genes (encoding form I RubisCO) of the cbbI operon. Juxtaposed between the genes encoding CbbR and CbbLS are genes that encode a three-protein two-component system (CbbRRS system) that functions to modify the ability of CbbR to regulate cbbLS expression. Previous studies indicated that the response regulators, as well as various coinducers (effectors), specifically influence CbbR-promoter interactions. In the current study, it was shown via several experimental approaches that the response regulators and coinducers act synergistically on CbbR to influence cbbLS transcription. Synergistic effects on the formation of specific CbbR-DNA complexes were quantified using surface plasmon resonance (SPR) procedures. Gel mobility shift and DNA footprint analyses further indicated structural changes in the DNA arising from the presence of response regulators and coinducer molecules binding to CbbR. Based on previous studies, and especially emphasized by the current investigation, it is clear that protein complexes influence promoter activity and the cbbLS transcription machinery.

Utilization of 5'-deoxy-nucleosides as Growth Substrates by Extraintestinal Pathogenic E. coli via the Dihydroxyacetone Phosphate Shunt.

All organisms utilize S-adenosyl-l-methionine (SAM) as a key co-substrate for methylation of biological molecules, synthesis of polyamines, and radical SAM reactions. When these processes occur, 5'-deoxy-nucleosides are formed as byproducts such as S-adenosyl-l-homocysteine (SAH), 5'-methylthioadenosine (MTA), and 5'-deoxyadenosine (5dAdo). One of the most prevalent pathways found in bacteria for the metabolism of MTA and 5dAdo is the DHAP shunt, which converts these compounds into dihydroxyacetone phosphate (DHAP) and 2-methylthioacetaldehyde or acetaldehyde, respectively. Previous work has shown that the DHAP shunt can enable methionine synthesis from MTA or serve as an MTA and 5dAdo detoxification pathway. Here we show that in Extraintestinal Pathogenic E. coil (ExPEC), the DHAP shunt serves none of these roles in any significant capacity, but rather physiologically functions as an assimilation pathway for use of MTA and 5dAdo as growth substrates. This is further supported by the observation that when MTA is the substrate for the ExPEC DHAP shunt, the sulfur components is not significantly recycled back to methionine, but rather accumulates as 2-methylthioethanol, which is slowly oxidized non-enzymatically under aerobic conditions. While the pathway is active both aerobically and anaerobically, it only supports aerobic ExPEC growth, suggesting that it primarily functions in oxygenic extraintestinal environments like blood and urine versus the predominantly anoxic gut. This reveals a heretofore overlooked role of the DHAP shunt in carbon assimilation and energy metabolism from ubiquitous SAM utilization byproducts and suggests a similar role may occur in other pathogenic and non-pathogenic bacteria with the DHAP shunt.

Further unraveling the regulatory twist by elucidating metabolic coinducer-mediated CbbR-cbbI promoter interactions in Rhodopseudomonas palustris CGA010.

The cbb(I) region of Rhodopseudomonas palustris (Rp. palustris) contains the cbbLS genes encoding form I ribulose-1,5-bisphosphate (RuBP) carboxylase oxygenase (RubisCO) along with a divergently transcribed regulator gene, cbbR. Juxtaposed between cbbR and cbbLS are the cbbRRS genes, encoding an unusual three-protein two-component (CbbRRS) system that modulates the ability of CbbR to influence cbbLS expression. The nature of the metabolic signals that Rp. palustris CbbR perceives to regulate cbbLS transcription is not known. Thus, in this study, the CbbR binding region was first mapped within the cbbLS promoter by the use of gel mobility shift assays and DNase I footprinting. In addition, potential metabolic coinducers (metabolites) were tested for their ability to alter the cbbLS promoter binding properties of CbbR. Gel mobility shift assays and surface plasmon resonance analyses together indicated that biosynthetic intermediates such as RuBP, ATP, fructose 1,6-bisphosphate, and NADPH enhanced DNA binding by CbbR. These coinducers did not yield identical CbbR-dependent DNase I footprints, indicating that the coinducers caused significant changes in DNA structure. These in vitro studies suggest that cellular signals such as fluctuating metabolite concentrations are perceived by and transduced to the cbbLS promoter via the master regulator CbbR.

Mechanistic diversity in the RuBisCO superfamily: RuBisCO from Rhodospirillum rubrum is not promiscuous for reactions catalyzed by RuBisCO-like proteins.

d-Ribulose 1,5-bisphosphate carboxylase/oxygenases (RuBisCOs) are promiscuous, catalyzing not only carboxylation and oxygenation of d-ribulose 1,5-bisphosphate but also other promiscuous, presumably nonphysiological, reactions initiated by abstraction of the 3-proton of d-ribulose 1,5-bisphosphate. Also, RuBisCO has homologues that do not catalyze carboxylation; these are designated RuBisCO-like proteins or RLPs. Members of the two families of RLPs catalyze reactions in the recycling of 5'-methylthioadenosine (MTA) generated by polyamine synthesis: (1) the 2,3-diketo-5-methylthiopentane 1-phosphate (DK-MTP 1-P) "enolase" reaction in the well-known "methionine salvage" pathway in Bacillus sp. and (2) the 5-methylthio-d-ribulose 1-phosphate (MTRu 1-P) 1,3-isomerase reaction in the recently discovered "MTA-isoprenoid shunt" that generates 1-deoxy-d-xylulose 5-phosphate for nonmevalonate isoprene synthesis in Rhodospirillum rubrum. We first studied the structure and reactivity of DK-MTP 1-P that was reported to decompose rapidly [Ashida, H., Saito, Y., Kojima, C., and Yokota, A. (2008) Biosci., Biotechnol., Biochem. 72, 959-967]. The 2-carbonyl group of DK-MTP 1-P is rapidly hydrated and can undergo enolization both nonenzymatically and enzymatically via the small amount of unhydrated material that is present. We then examined the ability of RuBisCO from R. rubrum to catalyze both of the RLP-catalyzed reactions. Contrary to a previous report [Ashida, H., Saito, Y., Kojima, C., Kobayashi, K., Ogasawara, N., and Yokota, A. (2003) Science 302, 286-290], we were unable to confirm that this RuBisCO catalyzes the DK-MTP 1-P "enolase" reaction either in vitro or in vivo. We also determined that this RuBisCO does not catalyze the MTRu 1-P 1,3-isomerase reaction in vitro. Thus, although RuBisCOs can be functionally promiscuous, RuBisCO from R. rubrum is not promiscuous for either of the known RLP-catalyzed reactions.

1-methylthio-D-xylulose 5-phosphate methylsulfurylase: a novel route to 1-deoxy-D-xylulose 5-phosphate in Rhodospirillum rubrum.

Rhodospirillum rubrum produces 5-methylthioadenosine (MTA) from S-adenosylmethionine in polyamine biosynthesis; however, R. rubrum lacks the classical methionine salvage pathway. Instead, MTA is converted to 5-methylthio-d-ribose 1-phosphate (MTR 1-P) and adenine; MTR 1-P is isomerized to 1-methylthio-d-xylulose 5-phosphate (MTXu 5-P) and reductively dethiomethylated to 1-deoxy-d-xylulose 5-phosphate (DXP), an intermediate in the nonmevalonate isoprenoid pathway [Erb, T. J., et al. (2012) Nat. Chem. Biol., in press]. Dethiomethylation, a novel route to DXP, is catalyzed by MTXu 5-P methylsulfurylase. An active site Cys displaces the enolate of DXP from MTXu 5-P, generating a methyl disulfide intermediate.

Unravelling the regulatory twist--regulation of CO2 fixation in Rhodopseudomonas palustris CGA010 mediated by atypical response regulator(s).

In Rhodopseudomonas palustris CGA010, the LysR type regulator, CbbR, specifically controls transcription of the cbbLS genes encoding form I RubisCO. Previous genetic and physiological studies had indicated that a unique two-component (CbbRRS) system influences CbbR-mediated cbbLS transcription under conditions where CO(2) is the sole carbon source. In this study, we have established direct protein-protein interactions between the response regulators of the CbbRRS system and CbbR, using a variety of techniques. The bacterial two-hybrid system established a specific interaction between CbbR and CbbRR1 (response regulator 1 of the CbbRRS system), confirmed in vitro by chemical cross-linking. In addition, both response regulators (CbbRR1 and CbbRR2) played distinct roles in influencing the CbbR-cbbLS promoter interactions in gel mobility shift assays. CbbRR1 increased the binding affinity of CbbR at the cbb(I) promoter three- to fivefold while CbbRR2 appeared to stabilize CbbR binding. Specific interactions were further supported by surface plasmon resonance (SPR) analyses. In total, the results suggested that both response regulators, with no discernible DNA-binding domains, must interact with CbbR to influence cbbLS expression. Thus the CbbRRS system provides an additional level of transcriptional control beyond CbbR alone, and appears to be significant for potentially fine-tuning cbbLS expression in Rps. palustris.