Chloroplast fructose-i ,6-bisphosphatase (Fru-P2-ase) is an essential enzyme in the photosynthetic pathway of carbon dioxide fixation into sugars. The properties of the chloroplast enzyme are clearly distinct from cytosolic gluconeogenic Fru-P2-ases. Light-dependent activation by way of a ferredoxin/thioredoxin system and insensitivity to AMP inhibition are distinctive characteristics of the chloroplast enzyme. However, the chloroplast enzyme shows a high degree of amino acid sequence similarity to gluconeogenic Fru-P2-ases. Sequence data reported for a total of 285 residues (=75% of the structure) of the spinach chloroplast enzyme reveals a 46% amino acid sequence identity with pig kidney Fru-P2-ase. We now report the amino acid sequence of a region consisting of 46 additional residues. This region is located near the middle of the primary structure of the enzyme and it includes a 16-residue insert not present in other Fru-P2-ases. This sequence insert has two cysteines separated by only 4 amino acid residues (Cys-Val-Val-Asn-Val-Cys), a characteristic feature of at least three other enzymes containing redox-active cysteines. It appears likely that this region of chloroplast Fru-P2-ase is involved in light-dependent activation.Light plays an important role in the regulation of several enzymes involved in the synthetic or carbon-reduction phase of photosynthesis and in related biochemical processes. In most cases, light produces the activation of several chloroplast enzymes that are essentially inactive in the dark. The in vivo regulation of all these enzymes involves the ferredoxin/ thioredoxin system, which comprises ferredoxin, thioredoxin f or m, and ferredoxin/thioredoxin reductase. One of these light-regulated enzymes is chloroplast fructose-1,6-bisphosphatase , the enzyme that catalyzes the conversion offructose 1,6-bisphosphate to fructose 6-phosphate. The light-mediated activation Fru-P2-ase occurs in i'ivo by way of a ferredoxin/thioredoxin f system that converts an inactive oxidized form of the enzyme into reduced active Fru-P2-ase (see refs. 1 and 2). The activation process can be mimicked in vitro by reduction of the enzyme with dithiothreitol (3). In both cases, the activation reaction involves the reduction of a critical disulfide bond in the enzyme. This activation mechanism is a characteristic of chloroplast Fru-P2-ase that distinguishes the chloroplast enzyme from cytosolic Fru-P2-ase (4,5). In addition, the chloroplast enzyme is not sensitive to AMP inhibition (6, 7), a property of all cytosolic gluconeogenic Fru-P2-ases (4). Nevertheless, the chloroplast enzyme shows a high degree of amino acid sequence similarity with gluconeogenic Fru-P2-ases that suggests a common evolutionary origin for all Fru-P2-ases in spite of their different functions and modes of regulation (8, 9). Amino acid sequence data reported for a total of 285 residues (-75% of the structure) of the spinach chloroplast enzyme (9, 10) reveals a 46% sequence identity with pig kidney Fru-P2-ase. These studies have disclo...

Chloroplast fructose-1,6-bisphosphatase (FbPase) is an essential enzyme in the photosynthetic pathway of carbon dioxide fixation into sugars. The properties of the chloroplast enzyme are clearly distinct from those of cytosolic gluconeogenic FbPases. Light-dependent activation via a ferredoxin/thioredoxin system and insensitivity to inhibition by AMP are unique characteristics of the chloroplast enzyme. However, preliminary amino acid sequence data (78 residues) have demonstrated that a significant degree of amino acid sequence similarity exists between spinach chloroplast and mammalian gluconeogenic fructose-1,6-bisphosphatase [Harrsch, P.B., Kim, Y., Fox, J.L., & Marcus, F. (1985) Biochem. Biophys. Res. Commun. 133, 520-526]. In the present study, we have identified two structural features of spinach chloroplast FbPase that appear to be common to all FbPases. These include (a) the presence of a protease-sensitive area located in a region equivalent to residues 51-71 of mammalian FbPases and (b) the recognition of two conserved histidine residues, equivalent to histidines-253 and -311 of the mammalian enzymes. In addition, we have obtained sequence information accounting for more than three-fourths of the primary structure of spinach chloroplast FbPase. The high degree of homology observed between the chloroplast enzyme and gluconeogenic FbPases suggests a common evolutionary origin for all fructose-1,6-bisphosphatases in spite of their different functions and modes of regulation.

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