Comparative amino acid sequence of fructose-1,6-bisphosphatases: identification of a region unique to the light-regulated chloroplast enzyme.

Abstract

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...

Thioredoxins: structure and function in plant cells

“…The cyanobacterial enzymes all show two conserved cysteine residues at positions 102 and 119, and, in addition, a third in position 18 {Synechocystis sp numbering). These residues are conspicuously absent in the the plant chloroplastic sequences which contain an insertion region of c. 17 amino acids with three cysteine residues (Cysl53, Cysl73 and Cysl78 in the pea sequence) (Marcus, Moberly & Latshaw, 1988;Raines et al, 1988). According to these sequence comparisons, the redox regulatory nature of cyanobacterial and chloroplast fructose-1,6-bisphosphatases does not have the same molecular origin.…”

Heterodimer formation between thioredoxin f and fructose 1,6‐bisphosphatase from spinach chloroplasts

“…In the spinach enzyme these cysteines, Cys-155, Cys-174 and Cys-179, are located on a regulatory loop structure [9] and based on early sequencing results the regulatory disul¢de was proposed between Cys-174 and Cys-179 [10]. Later site-directed mutagenesis experiments demonstrated the central role of Cys-153 in pea [11,12] and Cys-157 in rapeseed [13] FBPase for regulation.…”

cDNA and gene sequences of wheat chloroplast sedoheptulose‐1,7‐bisphosphatase reveal homology with fructose‐1,6‐bisphosphatases

“…One pair of cysteines is separated by four intervening residues (CGGTAC), a structural arrangement which is found for the redox-active groups of a number of enzymes, including mercuric reductase, glutathione reductase, lipoamide dehydrogenase (Fox and Walsh, 1983) and two ribonucleotide reductases (Lin et al, 1987). A subset of these, whch includes Fru(1 ,6)P2ase but not Sed(l,7)P2ase, also share greater similarity of the residues flanked by the cysteine pair (Marcus et al, 1988).…”

“…The wheat and spinach chloroplast Fru(1 ,6)P2ases have been shown to have a unique insertion sequence (at position Leu1 53 in the pig sequence), containing two cysteine residues which may be involved in the light regulation of these enzymes (Raines et al, 1988;Marcus et al, 1988). The alignment in Fig.…”

“…Molecular studies have resulted in extensive amino acid sequence information, including that of pig kidney (Marcus et al, 19821, the wheat (Raines et al, 1988) and spinach (Marcus et al, 1988) chloroplast enzymes, and also in the determination of the crystallographic structure of the pig kidney enzyme to a resolution of 0.28 nm (Ke et al, 1989a(Ke et al, , b, 1990(Ke et al, , 1991. In contrast to this, Sed(l,7)P2ase (like ribulose-1,s-bisphosphate carboxylase and phosphoribulokinase) is unique to the photosynthetic carbon-reduction cycle; consequently, much less information on the molecular characteristics of Sed(1 ,7)P2ase is available.…”