Characterization of Chlamydomonas Ribulose-1,5-bisphosphate carboxylase/oxygenase variants mutated at residues that are post-translationally modified.

BACKGROUND: Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the chloroplast enzyme that fixes CO2 in photosynthesis, but the enzyme also fixes O2, which leads to the wasteful photorespiratory pathway. If we better understand the structure-function relationship of the enzyme, we might be able to engineer improvements. When the crystal structure of Chlamydomonas Rubisco was solved, four new posttranslational modifications were observed which are not present in other species. The modifications were 4-hydroxylation of the conserved Pro-104 and 151 residues, and S-methylation of the variable Cys-256 and 369 residues, which are Phe-256 and Val-369 in land plants. Because the modifications were only observed in Chlamydomonas Rubisco, they might account for the differences in kinetic properties between the algal and plant enzymes. METHODS: Site-directed mutagenesis and chloroplast transformation have been used to test the essentiality of these modifications by replacing each of the residues with alanine (Ala). Biochemical analyses were done to determine the specificity factors and kinetic constants. RESULTS: Replacing the modified-residues in Chlamydomonas Rubisco affected the enzyme's catalytic activity. Substituting hydroxy-Pro-104 and methyl-Cys-256 with alanine influenced Rubisco catalysis. CONCLUSION: This is the first study on these posttranslationally-modified residues in Rubisco by genetic engineering. As these forms of modifications/regulation are not available in plants, the modified residues could be a means to modulate Rubisco activity. GENERAL SIGNIFICANCE: With a better understanding of Rubisco structure-function, we can define targets for improving the enzyme.

Daphnetin methylation stabilizes the activity of phosphoribulokinase in wheat during cold acclimation.

The methylation of daphnetin (7,8-dihydroxycoumarin) to its 8-methyl derivative is catalyzed by a wheat (Triticum aestivum L.) O-methyltransferase (TaOMT1). This enzyme is regulated by cold and photosystem II excitation pressure (plastid redox state). Here, we investigated the biological significance of this methylation and its potential role in modulating the activity of kinases in wheat. To identify the potential kinases that may interact with daphnetin in wheat, the soluble protein extract from aerial parts of cold-acclimated wheat was purified by DEAE-cellulose separation and affinity chromatography on a daphnetin derivative (7,8-dihydroxy-4-coumarin acetic acid)-EAH sepharose column. Mass spectrometric analysis indicated that wheat phosphoribulokinase (TaPRK) is the major kinase that binds to daphnetin. This TaPRK plays an important role in regulating the flow of carbon through the Calvin cycle, by catalyzing the final step in the regeneration of ribulose 1,5-bisphosphate from ribulose-5-phosphate (Ru5P) and ATP. The activities of TaPRK, endogenous or recombinant, are inhibited by daphnetin in a specific and dose-dependent manner, but not by its monomethyl derivative (7-methyl, 8-hydroxycoumarin). Furthermore, HPLC-MS analysis of wheat extracts reveals that 7,8-dimethoxycoumarin is more abundant than its monomethyl derivative. The results also show that cold acclimation does not alter the level of TaPRK mRNA or its enzyme activity, and thus ensures the stable generation of ribulose 1,5-biphosphate.

Assimilation of formaldehyde in transgenic plants due to the introduction of the bacterial ribulose monophosphate pathway genes.

Formaldehyde (HCHO) is an air pollutant suspected of being carcinogenic and a cause of sick-house syndrome. Microorganisms called methylotrophs, which can utilize reduced C(1) compounds such as methane and methanol, fix and assimilate HCHO, whereas most plants are unable to assimilate HCHO directly. We found that a bacterial formaldehyde-fixing pathway (ribulose monophosphate pathway) can be integrated as a bypass to the Calvin-Benson cycle in transgenic Arabidopsis thaliana and tobacco by genetic engineering. These plants showed enhanced tolerance to HCHO and enhanced capacity to eliminate gaseous HCHO by fixing it as a sugar phosphate. Our results provide a novel strategy for phytoremediation of HCHO pollution, and also represent the first step toward the production of plants that can assimilate natural gas-derived C(1) compounds.

Development of nucleic acid isolation by non-silica-based nanoparticles and real-time PCR kit for edible vegetable oil traceability.

For efficient extraction of amplifiable DNA from edible vegetable oils, we developed a novel DNA extraction approach based on the non-silica-based dipolar nanocomposites. The nanoparticle comprises a hydrophilic polymethyl methacrylate core with abundant capillaries, hydrophilic vesicles decorated with molecules having DNA affinity and a coating hydrophobic polystyrene layer. The nanoparticles are soluble in oil, adsorb the DNA from the aqueous phase and gave a high DNA recovery ratio. All DNA extracts from fully refined vegetable oil soybean, peanut, rapeseed, and cottonseed oils, including their blends, were sufficiently pure to be amplified by real-time PCR targeting the chloroplast ribulose-1,5-bisphosphate gene (rbcL), therefore, the species of origin and their ratios in mixed vegetable oils blended from two or three oil-species could be determined. These results indicate that the novel DNA isolation and real-time PCR kit is a simple, sensitive and efficient tool for the species identification and traceability in refined vegetable oils.

Structural Characterization of a Newly Identified Component of α-Carboxysomes: The AAA+ Domain Protein CsoCbbQ.

Carboxysomes are bacterial microcompartments that enhance carbon fixation by concentrating ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and its substrate CO2 within a proteinaceous shell. They are found in all cyanobacteria, some purple photoautotrophs and many chemoautotrophic bacteria. Carboxysomes consist of a protein shell that encapsulates several hundred molecules of RuBisCO, and contain carbonic anhydrase and other accessory proteins. Genes coding for carboxysome shell components and the encapsulated proteins are typically found together in an operon. The α-carboxysome operon is embedded in a cluster of additional, conserved genes that are presumably related to its function. In many chemoautotrophs, products of the expanded carboxysome locus include CbbO and CbbQ, a member of the AAA+ domain superfamily. We bioinformatically identified subtypes of CbbQ proteins and show that their genes frequently co-occur with both Form IA and Form II RuBisCO. The α-carboxysome-associated ortholog, CsoCbbQ, from Halothiobacillus neapolitanus forms a hexamer in solution and hydrolyzes ATP. The crystal structure shows that CsoCbbQ is a hexamer of the typical AAA+ domain; the additional C-terminal domain, diagnostic of the CbbQ subfamily, structurally fills the inter-monomer gaps, resulting in a distinctly hexagonal shape. We show that CsoCbbQ interacts with CsoCbbO and is a component of the carboxysome shell, the first example of ATPase activity associated with a bacterial microcompartment.

Organization of the genes involved in the ribulose monophosphate pathway in an obligate methylotrophic bacterium, Methylomonas aminofaciens 77a.

The 4.4-kb PstI fragment harboring the gene encoding 3-hexulose-6-phosphate synthase, rmpA, which was previously cloned from the chromosome of an obligate methylotroph, Methylomonas aminofaciens 77a, was investigated in detail. In addition to the rmpA gene, the fragment contained three open reading frames encoding transaldolase (rmpD), IS10-R (rmpI), and 6-phospho-3-hexuloisomerase (PHI) (rmpB). The rmpB gene product was overproduced in Escherichia coli cells, purified to homogeneity, and then enzymatically identified as PHI. The gene organization of the ribulose monophosphate pathway enzymes together with a transposon, IS10-R, is discussed from both evolutionary and regulatory aspects.

Novel sucrose-dependent adhesion co-factors in Streptococcus mutans.

Streptococcus mutans glucosyltransferases form extracellular glucans from sucrose to promote adhesion to the teeth. We tested whether additional factors are involved in S. mutans sucrose-dependent adhesion. By screening a pVA891-insertion mutant library of S. mutans LT11, we isolated four clones deficient in adhesion to glass in the presence of sucrose, but normal in glucosyltransferase activities. The genetic loci flanking the insertion sites were retrieved and identified. They encode glycerol-3-phosphate dehydrogenase, an ABC transporter, a multidrug-efflux pump, and either the ribulose monophosphate operon or ascorbate metabolism operon. The four mutants were analyzed for their phenotypic expression and in vivo colonization in rats. The multidrug efflux pump mutant failed to colonize the rats. Three other mutants colonized the rats by reverting to the wild type. Therefore, these four factors may contribute to S. mutans sucrose-dependent adhesion.

Codon usage in plant genes.

We have examined codon bias in 207 plant gene sequences collected from Genbank and the literature. When this sample was further divided into 53 monocot and 154 dicot genes, the pattern of relative use of synonymous codons was shown to differ between these taxonomic groups, primarily in the use of G + C in the degenerate third base. Maize and soybean codon bias were examined separately and followed the monocot and dicot codon usage patterns respectively. Codon preference in ribulose 1,5 bisphosphate and chlorophyll a/b binding protein, two of the most abundant proteins in leaves was investigated. These highly expressed are more restricted in their codon usage than plant genes in general.

Processing of chimeric introns in dicot plants: evidence for a close cooperation between 5' and 3' splice sites.

Splice sites of vertebrate introns are generally not recognized in plant cells. Several lines of evidences have led to the proposal that the mechanism of 3' splice site selection differs in plants and animals (K. Wiebauer, J.J. Herrero, and W. Filipowicz, Mol. Cell. Biol. 8:2042-2051, 1988). To gain a better insight into the mechanistic differences between plant and animal splicing, we constructed chimeric introns consisting partly of dicotyledonous plant and partly of animal intron sequences. Splicing of these chimeric introns was analyzed in transiently transfected tobacco protoplasts. The results show that there are no principal sequence or structural differences between the 3' splice regions of plants and animals. Furthermore, evidence is provided that cooperation between 5' and 3' splice sites takes place and influences their mutual selection.

Bacillus subtilis yckG and yckF encode two key enzymes of the ribulose monophosphate pathway used by methylotrophs, and yckH is required for their expression.

The ribulose monophosphate (RuMP) pathway is one of the metabolic pathways for the synthesis of compounds containing carbon-carbon bonds from one-carbon units and is found in many methane- and methanol-utilizing bacteria, which are known as methylotrophs. The characteristic enzymes of this pathway are 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI), neither of which was thought to exist outside methylotrophs. However, the presumed yckG gene product (YckG) of Bacillus subtilis shows a primary structure similar to that of methylotroph HPS (F. Kunst et al., Nature 390:249-256, 1997). We have also investigated the sequence similarity between the yckF gene product (YckF) and methylotroph PHI (Y. Sakai, R. Mitsui, Y. Katayama, H. Yanase, and N. Kato, FEMS Microbiol. Lett. 176:125-130, 1999) and found that the yckG and yckF genes of B. subtilis express enzymatic activities of HPS and PHI, respectively. Both of these activities were concomitantly induced in B. subtilis by formaldehyde, with induction showing dependence on the yckH gene, but were not induced by methanol, formate, or methylamine. Disruption of either gene caused moderate sensitivity to formaldehyde, suggesting that these enzymes may act as a detoxification system for formaldehyde in B. subtilis. In conclusion, we found an active yckG (for HPS)-yckF (for PHI) gene structure (now named hxlA-hxlB) in a nonmethylotroph, B. subtilis, which inherently preserves the RuMP pathway.

A novel operon encoding formaldehyde fixation: the ribulose monophosphate pathway in the gram-positive facultative methylotrophic bacterium Mycobacterium gastri MB19.

A 4.2-kb PstI fragment harboring the gene cluster of the ribulose monophosphate (RuMP) pathway for formaldehyde fixation was identified in the chromosome of a gram-positive, facultative methylotroph, Mycobacterium gastri MB19, by using the coding region of 3-hexulose-6-phosphate synthase (HPS) as the hybridization probe. The PstI fragment contained three complete open reading frames (ORFs) which encoded from the 5' end, a DNA-binding regulatory protein (rmpR), 6-phospho-3-hexuloisomerase (PHI; rmpB), and HPS (rmpA). Sequence analysis suggested that rmpA and rmpB constitute an operon, and Northern blot analysis of RNA extracted from bacteria grown under various conditions suggested that the expression of the two genes is similarly regulated at the transcriptional level. A similarity search revealed that the proteins encoded by rmpA and rmpB in M. gastri MB19 show high similarity to the unidentified proteins of nonmethylotrophic prokaryotes, including bacteria and anaerobic archaea. The clusters in the phylogenetic tree of the HPS protein of M. gastri MB19 and those in the phylogenetic tree of the PHI protein were nearly identical, which implies that these two formaldehyde-fixing genes evolved as a pair. These findings give new insight into the acquisition of the formaldehyde fixation pathway during the evolution of diverse microorganisms.