Structure and Activity of the Metal-independent Fructose-1,6-bisphosphatase YK23 from Saccharomyces cerevisiae

Abstract

Fructose-1,6-bisphosphatase (FBPase), a key enzyme of gluconeogenesis and photosynthetic CO 2 fixation, catalyzes the hydrolysis of fructose 1,6-bisphosphate (FBP) to produce fructose 6-phosphate, an important precursor in various biosynthetic pathways. All known FBPases are metal-dependent enzymes, which are classified into five different classes based on their amino acid sequences. Eukaryotes are known to contain only the type-I FBPases, whereas all five types exist in various combinations in prokaryotes. Here we demonstrate that the uncharacterized protein YK23 from Saccharomyces cerevisiae efficiently hydrolyzes FBP in a metal-independent reaction. YK23 is a member of the histidine phosphatase (phosphoglyceromutase) superfamily with homologues found in all organisms. The crystal structure of the YK23 apo-form was solved at Fructose-1,6-bisphosphatase (FBPase) 2 (EC 3.1.3.11) catalyzes the removal of phosphate 1 from fructose 1,6-bisphosphate to produce fructose 6-phosphate, an important precursor in various biosynthetic pathways. FBPase is a key, rate-controlling enzyme of gluconeogenesis, an important metabolic pathway that allows the cells to synthesize glucose and grow on non-carbohydrate carbon sources, such as glycerol, organic acids, and amino acids (1). Gluconeogenesis is also responsible for excessive glucose production found in type 2 diabetes, which is a growing worldwide health concern (2). Because FBPases represent the major control point and function only in gluconeogenesis in mammals, they are recognized as an attractive target for the development of drugs for the treatment of type 2 diabetes (3). FBPase also functions at the branch point between the regenerative phase of the photosynthetic CO 2 fixation cycle (Calvin), which is the primary pathway of carbon fixation, and the starch biosynthesis (4). In addition, FBPase is required for virulence in Mycobacterium tuberculosis and Leishmania major and plays an important role in the production of lysine and glutamate by Corynebacterium glutamicum (5, 6).Most characterized FBPases belong to the superfamily of lithium-sensitive metal-dependent phosphatases that also includes three families of inositol phosphatases (clan CL0171, Pfam data base) (7). Based on the amino acid sequence, FBPases can be assigned to one of the five proposed classes (8 -10). All known FBPases require a divalent metal cation for activity (Mg 2ϩ or Mn 2ϩ ), are inhibited by Li ϩ , and their activity is often regulated by AMP, fructose 2,6-bisphosphate, and phosphoenolpyruvate (11-13). Types I, II, and IV FBPases and inositol monophosphatases have similar three-dimensional structures with a sugar phosphatase-fold (␣␤␣␤␣) suggesting a common evolutionary origin and catalytic mechanism (14 -16). All five types of FBPases are normally found in prokaryotes, where types I, II, and III are more common to bacteria (8), type IV are mostly found in archaea (10), and type V in thermophiles from both domains (9). The majority of organisms have more than one FBPase, mostly the combinatio...

Proteomic analysis of excretory secretory products from Clonorchis sinensis adult worms: molecular characterization and serological reactivity of a excretory–secretory antigen-fructose-1,6-bisphosphatase

“…When C. sinensis resides in bile ducts and causes clonorchiasis inducing hepatic fibrosis, carbohydrates such as glucose and glycogen are the main energy source for its metabolism. FBPase catalyzes the hydrolysis of fructose 1,6-bisphosphate (FBP) to fructose 6-phosphate, which is an important precursor in several biosynthetic pathways (Kuznetsova et al 2010). To determine whether Cs-FBPase may be a candidate pathogenic ESP, we performed several molecular-and immunology-based analyses.…”

Structure and activity of the Saccharomyces cerevisiae dUTP pyrophosphatase DUT1, an essential housekeeping enzyme

“…The modified pET15b vector contains an N-terminal His 6 tag followed by a tobacco etch virus protease cleavage site (ENLYFQ↓;G) [18]. DUT1 was overexpressed in the E. coli BL21-Gold(DE3) strain (Stratagene) and affinity purified using Ni 2+ -chelate chromatography on nickel-affinity resin (Qiagen) as described previously [18]. The oligomeric state of DUT1 was determined using gel-filtration analysis on a Superdex-200 10/300 GL column (GE Healthcare) as described previously [18].…”

Functional Diversity of Haloacid Dehalogenase Superfamily Phosphatases from Saccharomyces cerevisiae

“…Purification of proteins for screening and biochemical characterization was performed as described previously (48). The oligomeric state of purified proteins was determined using gel filtration analysis on a Superdex 200 10/300 column (GE Healthcare).…”

“…The oligomeric state of purified proteins was determined using gel filtration analysis on a Superdex 200 10/300 column (GE Healthcare). Site-directed mutagenesis of YKR070W was performed using a protocol based on the QuikChange site-directed mutagenesis kit (Stratagene) as described previously (48). The presence of mutations was verified by DNA sequencing, and the mutant proteins were overexpressed and purified in the same manner as the wild-type YKR070W.…”

“…Secondary phosphatase screens with 93 phosphorylated metabolites (supplemental Table 2) were then performed to identify the preferred in vitro substrates for these proteins (40). Phosphatase activity against phosphorylated metabolites and phosphopeptides was measured spectrophotometrically using the malachite green reagent or a mild phosphate detection method (48). The dependence of phosphatase activity on divalent metal cations was determined using saturating concentrations of the indicated substrates and cations (5 mM Mg 2ϩ or 0.5 mM for other ions), whereas the pH dependence was determined using a mixed buffer system (MEGA buffer) (49).…”