Expression, Purification, and Characterization of Natural Mutants of Human Aldolase B

Abstract

Fructaldolases (EC 4.1.2.13) are ancient enzymes of glycolysis that catalyze the reversible cleavage of phosphofructose esters into cognate triose (phosphates). Three vertebrate isozymes of Class I aldolase have arisen by gene duplication and display distinct activity profiles with fructose 1,6-bisphosphate and with fructose 1-phosphate. We describe the biochemical and biophysical characterization of seven natural human aldolase B variants, identified in patients suffering from hereditary fructose intolerance and expressed as recombinant proteins in E. coli, from which they were purified to homogeneity. The mutant aldolases were all missense variants and could be classified into two principal groups: catalytic mutants, with retained tetrameric structure but altered kinetic properties (W147R, R303W, and A337V), and structural mutants, in which the homotetramers readily dissociate into subunits with greatly impaired enzymatic activity (A149P, A174D, L256P, and N334K). Investigation of these two classes of mutant enzyme suggests that the integrity of the quaternary structure of aldolase B is critical for maintaining its full catalytic function.

Inhibition of Nicotinamide Phosphoribosyltransferase (NAMPT), an Enzyme Essential for NAD+ Biosynthesis, Leads to Altered Carbohydrate Metabolism in Cancer Cells

“…Third, all three human aldolase isozymes are known to convert dihydroxyacetone phosphate and glyceraldehyde to fructose 1-phosphate (30 -32). In addition, a rabbit aldolase was shown to convert dihydroxyacetone phosphate and erythrose to sedoheptulose 1-phosphate (30), and this finding has been confirmed in this study. Furthermore, the labeling and biochemical studies showed that the rate of sedoheptulose 1-phosphate synthesis was much slower than that of fructose 1-phosphate, consistent with their cellular levels (Figs.…”

Hereditary fructose intolerance: functional study of two novel ALDOB natural variants and characterization of a partial gene deletion

“…In addition, to evaluate metabolic effects of the novel ALDOB variants also in actual cells, we now start cloning ALDOB cDNA in a eukaryotic expression system. Models of natural variants based on crystal structural data may also shed light on mutationinduced structural-functional alterations [Santamaria et al, 2000;Rellos et al, 2000;Esposito et al, 2002;Esposito et al, 2004a;Malay et al, 2005]. Analysis of the ALDOB three-dimensional structures clearly indicates that both Arg46 and Tyr343 are exposed to the solvent, far from the tetramer interface ( Arg 304 in the correct position for substrate binding and catalysis (Fig.…”

“…The effects of various HFI-related missense mutations on ALDOB function have been investigated by in vitro expression and functional characterization of the corresponding recombinant enzymes [Brooks and Tolan, 1994;Santamaria et al, 2000;Rellos et al, 2000;Esposito et al, 2002;Esposito et al, 2004a]. Thus, to assess whether the p.R46W and p.Y343H natural ALDOB variants are associated to a functional deficiency, we expressed the corresponding recombinant enzymes in bacterial cells.…”

Glycolytic and Non-glycolytic Functions of Mycobacterium tuberculosis Fructose-1,6-bisphosphate Aldolase, an Essential Enzyme Produced by Replicating and Non-replicating Bacilli

“…An untagged form of FBA-tb was produced because N-terminal tags have been found to affect dramatically the solubility and activity of FBA-tb produced in E. coli probably by disrupting its quaternary structure (18,49). Moreover, we found a C-terminal hexahistidinetagged form of FBA-tb to be inactive when expressed in M.…”