Primary structure and phylogeny of the Calvin cycle enzymes transketolase and fructosebisphosphate aldolase of Xanthobacter flavus

Abstract

Xanthobacter flavus grows autotrophically by using the Calvin cycle for the fixation of CO 2 . Only 2 of the 11 enzymes of the Calvin cycle are characteristic for this pathway; the others are also present during heterotrophic growth. The key enzymes of the Calvin cycle, phosphoribulokinase and ribulosebisphosphate carboxylase, are encoded within the cbb operon, which is transcribed only during autotrophic growth. Two additional genes are located within this operon: cbbX, encoding a protein with unknown function, and cbbF, encoding fructosebisphosphatase (27,29). The transcription of the cbb operon is positively regulated by CbbR, a LysR-type transcriptional regulator, which binds to two sites in the cbb promoter (47).During autotrophic growth, X. flavus uses two fructosebisphosphatase enzymes with distinct properties. The inducible enzyme encoded by cbbF has a high level of sedoheptulosebisphosphatase activity and is stimulated by ATP. The second constitutive fructosebisphosphatase has a low level of sedoheptulosebisphosphatase activity and is not stimulated by ATP (48). In contrast to the fructosebisphosphatase isoenzyme pair, only one phoshoglycerate kinase gene, which is not encoded within the cbb operon, is employed by X. flavus. The pgk gene is constitutively expressed, but the expression level is higher during autotrophic growth than during heterotrophic growth (26).Little is known about the transketolase (EC 2.2.1.1) and fructosebisphosphate aldolase (FBP aldolase; EC 4.1.2.13) enzymes of X. flavus. Like fructosebisphosphatase and phosphoglycerate kinase, these enzymes are involved in both heterotrophic metabolism and the fixation of CO 2 via the Calvin cycle. Two unrelated mechanistically distinct types of FBP aldolase enzymes are encountered in bacteria, archaea, and eukarya (24). Class I FBP aldolases form a covalent Schiff base between the substrate and the ␣-amino group of a lysine residue during catalysis, whereas the class II enzymes depend on a divalent cation as the electrophile in the catalytic cycle (24).X. flavus (Table 1) was grown heterotrophically on succinate (10 mM) or gluconate (10 mM) and autotrophically on methanol (0.5% [vol/vol]) at 30ЊC as described previously (22,28). The activities of transketolase and FBP aldolase were determined, according to published methods (15, 49), in cell extracts which were prepared by using a French pressure cell as described previously (27). The activity of transketolase was increased sixfold following autotrophic growth on methanol compared with that of heterotrophically grown cells. In sharp contrast, the activity of FBP aldolase (without Fe 2ϩ ) was the same for both heterotrophic and autotrophic growth. Because the activity of class II FBP aldolase is dependent on Fe 2ϩ as the electrophile (24), FeSO 4 (700 M) was included in the reaction assay. Surprisingly, Fe 2ϩ did not affect the FBP aldolase activity in the cell extracts of heterotrophically grown cells but stimulated the activity of FBP aldolase in cell extracts of autotrophically grown cells ...

Molecular Evolutionary Analysis of the Thiamine-Diphosphate-Dependent Enzyme, Transketolase

“…7) clustered the ␥ subdivision with the plants and yeasts. A similar result was reported for the class II FBP aldolase (Van Den Bergh et al 1996).…”

“…All four organisms belong to the ␣ and ␤ subdivisions of the proteobacteria. The same result has been reported using the sequences of the class II fructosebisphosphate (FBP) aldolase (Van Den Bergh et al 1996). In contrast, the two NJ trees (Figs.…”

“…All 19 phylogenetic trees generated by various methods (e.g., indicate that the branching order obtained from transketolase sequences is, in general, conventional, as has been reported elsewhere (Van Den Bergh et al 1996). There is a distinct subdivision into four groups representing mammals, yeasts, bacteria, and plants, as would be expected from their taxonomic relationship.…”

“…Given the range of sequences now available for study, it is possible to attempt the phylogenetic reconstruction of this family. Several reports have inferred the phylogeny of transketolase on the basis of dendograms calculated from relatively few sequences (Reizer et al 1993;Sundström et al 1993;Van Den Bergh et al 1996), providing an interesting but limited insight into the evolution of this enzyme.…”

Ribulose bisphosphate carboxylase activity and a Calvin cycle gene cluster in Sulfobacillus species

“…3). A suggestion of a Gram-positive bacterium as a possible origin of a CBB-active enzyme of some proteobacteria arose previously from the apparent relatedness of their CbbA to the class II FBP aldolase of B. subtilis (van den Bergh et al, 1996). However, the RuBisCO of S. acidophilus shows no closer relationship than other red-type RuBisCOs to the distantly related RuBisCO-like proteins (including examples in several Bacillus species) which could be closer to a common ancestor of CBB cycle-active RuBisCOs (Ashida et al, 2005), giving no support for particularly early evolution of a bacterial CBB cycle in Sulfobacillus-like species.…”