THE FORM-II FRUCTOSE 1,6-BISPHOSPHATASE AND PHOSPHORIBULOKINASE GENES FORM PART OF A LARGE OPERON IN RHODOBACTER-SPHAEROIDES - PRIMARY STRUCTURE AND INSERTIONAL MUTAGENESIS ANALYSIS

Fructose 1,6-bisphosphatase (FBPase) and phosphoribulokinase (PRK) are two key enzymes of the reductive pentose phosphate pathway or Calvin cycle of photosynthetic carbon dioxide assimilation. Early studies had indicated that the properties of enzymes isolated from photosynthetic bacteria were clearly distinct from those of enzymes obtained from the chloroplasts of higher plants [for a review, see Tabita (1988)]. The eucaryotic enzymes, which are light activated by the thioredoxin/ferredoxin system (Buchanan, 1980), were each shown to contain a putative regulatory amino acid sequence (Marcus et al., 1988; Porter et al., 1988). The enzymes from photosynthetic bacteria are not controlled by the thioredoxin/ferredoxin system but exhibit complex kinetic properties and, in the case of PRK, there is an absolute requirement of NADH for activity. In the photosynthetic bacterium Rhodobacter sphaeroides, the structural genes of the Calvin cycle, including the genes that encode FBPase (fbp) and PRK (prk), are found in two distinct clusters, and the fbp and prk genes are closely associated in each cluster. In the present investigation, we have determined the nucleotide sequence of the fbpB and prkB genes of the form II cluster and have compared the deduced amino acid sequences to previously determined sequences of light-activated enzymes from higher plants and from other eucaryotic and procaryotic sources. In the case of FBPase, there are several regions that are conserved in the R. sphaeroides enzymes, including a protease-sensitive area located in a region equivalent to residues 51–71 of mammalian FBPase. There are also two conserved histidine residues that are separated by 56–59 amino acids in all sources of FBPase heretofore examined, suggesting an important structural role. The sequence required for light activation of the chloroplast enzyme is not present in the R. sphaeroides FBPase nor is the AMP-sensitive region of the mammalian enzyme. For PRK, the lack of overall homology to the plant enzyme is striking, yet there are three regions that exhibit significant similarities, including a putative ATP-binding site. Evidence is also presented that strongly suggests that the fbpB and prkB genes are cotranscribed and part of an operon with several other CÓ2 assimilatory structural genes. © 1990, American Chemical Society. All rights reserved.