Structure of E69Q mutant of human muscle fructose-1,6-bisphosphatase

Abstract

Human fructose-1,6-bisphosphatase is an allosteric enzyme that is regulated by different ligands. There are only two known isozymes in human tissues: the liver isozyme (the key enzyme of gluconeogenesis), which is regulated by fructose 2,6-bisphosphate, and its muscle counterpart (participating in glycogen synthesis), which is regulated by calcium ions. AMP, which is an allosteric inhibitor of both isozymes, inhibits the muscle isozyme with an I(0.5) that is 35-100 times lower than for the liver isozyme and the reason for this difference remains obscure. In studies aiming at an explanation of the main differences in the regulation of the two isozymes, it has been shown that only one residue, in position 69, regulates the sensitivity towards calcium ions. As a consequence of this finding, an E69Q mutant of the muscle isozyme, which is insensitive to calcium ions while retaining all other kinetic properties resembling the liver isozyme, has been prepared and crystallized. Here, two crystal structures of this mutant enzyme in complex with AMP with and without fructose 6-phosphate (the product of the catalytic reaction) are presented. The AMP binding pattern of the muscle isozyme is quite similar to that of the liver isozyme and the T conformations of the two isozymes are nearly the same.

Crystal Structures of Human Muscle Fructose-1,6-Bisphosphatase: Novel Quaternary States, Enhanced AMP Affinity, and Allosteric Signal Transmission Pathway

“…The hmFru-1,6-Pase crystals in this report are in space group P 4 2 2 1 2 (X-ray data collection and refinement statistics for all structures are shown in Table 2), which is different from all the previously reported structures of Fru-1,6-Pases, including the AMP-bound T-state hmFru-1,6-Pase that was recently reported [31]. The crystals contain one subunit in each asymmetric unit.…”

“…Based on the electron density maps, the F6P molecule is present as the β-anomer, which differs from the α-anomer observed in the recently published T-state hmFru-1,6-Pase structure [31]. Notably, in accord with the slight movement of the side chain of Asp121, M1 is located 0.7 Å away from M1 in the wild-type structure, and is 1.7 Å distant from M1 in the product complexes of porcine Fru-1,6-Pase (PDB codes 1EYI and 1CNQ).…”

“…However, it should be noted that the conformation of the hinge (residues 51–54) in the Q32R mutant is the same as that in the engaged conformation for wild-type hmFru-1,6-Pase, but different from the disengaged conformation observed for the T-state hmFru-1,6-Pase [31] (PDB 3IFA). Other than this difference, superposition of the wild-type and Q32R mutant structures revealed that this variation induces significant conformational adjustments of the α1 and α2 helices (Figure 2c).…”

“…Since the mutant displayed interesting structural and kinetic properties, we then re-cloned the wild-type gene followed by structure-function studies. During the preparation of this manuscript, a paper describing the AMP-bound T-state structure of the hmFru-1,6-Pase E69Q mutant was published, in which a T-state structure, similar to other AMP-bound Fru-1,6-Pases, was described [31]. However, the mechanism underlying higher sensitivity towards AMP was not addressed in this recent paper.…”

In silico validation of microalgal metabolites against Diabetes mellitus

“…The activities of these enzymes enhanced production of glucose through glycerol or gluconeogenic amino acids [14]. The constant formation of glucose affected serious non-insulin dependent diabetic conditions.…”