Molecular and biochemical characterization of a distinct type of fructose-1,6-bisphosphatase from Pyrococcus furiosus

Abstract

The Pyrococcus furiosus fbpA gene was cloned and expressed in Escherichia coli, and the fructose-1,6-bisphosphatase produced was subsequently purified and characterized. The dimeric enzyme showed a preference for fructose-1,6-bisphosphate, with a K(m) of 0.32 mM and a V(max) of 12.2 U/mg. The P. furiosus fructose-1,6-bisphosphatase was strongly inhibited by Li(+) (50% inhibitory concentration, 1 mM). Based on the presence of conserved sequence motifs and the substrate specificity of the P. furiosus fructose-1,6-bisphosphatase, we propose that this enzyme belongs to a new family, class IV fructose-1,6-bisphosphatase.

FIG. 1.

Multiple sequence alignment of the deduced amino acid sequence of the P. furiosus FBPase with the sequences of its FBPase IV homologs and of I-1-Pases and FBPases from eukarya and bacteria. Abbreviations (accession numbers are given in parentheses): H.s. IMP, Homo sapiens I-1-Pase 1 (P29218); E.c. IMP, E. coli SuhB I-1-Pase (P22783); T.m. FBPIV, T. maritima TM1415 FBPase (O33832); A.f. FBPIV; A. fulgidus AF2372 FBPase (NP_071195); M.j. FBPIV, M. jannaschii MJ0109 FBPase (Q57573); P.f. FBPIV, P. furiosus FBPase (AF453319); E.c. FBP1, E. coli FBPase (P09200); S.s. FBP, Sus scrofa FBPase (P00636). Gaps introduced by the alignment are indicated by dashes. Completely conserved regions are indicated by black boxes. Highly conserved regions are shaded gray. The IMP motifs are indicated with horizontal black bars above the alignments. The FBPase motif is indicated with a horizontal gray bar under the alignment. IMP 1 motif, [FWV]-x (0, 1)-[LIVM]-D-P-[LIVM]-D-[SG]-[ST]-x (2)-[FY]-x-[HKRNSTY]; inositol monophosphatase family signature 1 (PS00629). IMP 2 motif, [WV]-D-x-[AC]-[GSA]-[GSAPV]-x-[LIVACP]-[LIV]-[LIVAC]-x (3)-[GH]-[GA]; inositol monophosphatase family signature 2 (PS00630). FBPase motif, [AG]-[RK]-[LI]-x (1, 2)-[LIV]-[FY]-E-x (2)-P-[LIVM]-[GSA] (PS00124) (http://www.expasy.ch/prosite). Asterisks denote residues involved in the Li⁺ binding site (24). The determined N-terminal amino acid sequence from the purified P. furiosus FBPase described in the text is underlined.