Expression, purification, and characterization of natural mutants of human aldolase B. Role of quaternary structure in catalysis.

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.

Alteration of substrate specificity by a naturally-occurring aldolase B mutation (Ala337-->Val) in fructose intolerance.

A molecular analysis of human aldolase B genes in two newborn infants and a 4-year-old child with hereditary fructose intolerance, the offspring of a consanguineous union, has identified the novel mutation Ala337-->Val in homozygous form. This mutation was also detected independently in two other affected individuals who were compound heterozygotes for the prevalent aldolase B allele, Ala149-->Pro, indicating that the mutation causes aldolase B deficiency. To test for the effect of the mutation, catalytically active wild-type human aldolase B and the Val337 variant enzyme were expressed in Escherichia coli. The specific activities of the wild-type recombinant enzyme were 4.8 units/mg and 4.5 units/mg towards fructose 1,6-bisphosphate (FBP) and fructose 1-phosphate (F-1-P) as substrates with Michaelis constants of 4 microM and 2.4 mM respectively. The specific activities of purified tetrameric Val337 aldolase B, which affects an invariant residue in the C-terminal region, were 4.2 units/mg and 2.6 units/mg towards FBP and F-1-P as substrates respectively; the corresponding Michaelis constants were 22 microM and 24 mM. The FBP-to-F-1-P substrate activity ratios were 0.98 and 1.63 for wild-type and Val337 variant enzymes respectively. The Val337 mutant aldolase had an increased susceptibility to proteolytic cleavage in E. coli and rapidly lost activity on storage. Comparative CD determinations showed that the Val337 protein had a distinct thermal denaturation profile with markedly decreased enthalpy, indicating that the mutant protein is partly unfolded. The undegraded mutant had preferentially decreased affinity and activity towards its specific F-1-P substrate and maintained appreciable activity towards FBP. In contrast, fluorescence studies of the mutant showed an increased binding affinity for products of the aldolase reaction, indicating a role for the C-terminus in mediating product release. These findings in a rare but widespread naturally occurring mutant implicate the C-terminus in the activity of human aldolase B towards its specific substrates and demonstrate its role in maintaining the overall stability of the enzyme tetramer.

Hereditary fructose intolerance.

Hereditary fructose intolerance (HFI, OMIM 22960), caused by catalytic deficiency of aldolase B (fructose-1,6-bisphosphate aldolase, EC 4.1.2.13), is a recessively inherited condition in which affected homozygotes develop hypoglycaemic and severe abdominal symptoms after taking foods containing fructose and cognate sugars. Continued ingestion of noxious sugars leads to hepatic and renal injury and growth retardation; parenteral administration of fructose or sorbitol may be fatal. Direct detection of a few mutations in the human aldolase B gene on chromosome 9q facilitates the genetic diagnosis of HFI in many symptomatic patients. The severity of the disease phenotype appears to be independent of the nature of the aldolase B gene mutations so far identified. It appears that hitherto there has been little, if any, selection against mutant aldolase B alleles in the population: in the UK, approximately 1.3% of neonates harbour one copy of the prevalent A149P disease allele. The ascendance of sugar as a major dietary nutrient, especially in western societies, may account for the increasing recognition of HFI as a nutritional disease and has shown the prevalence of mutant aldolase B genes in the general population. The severity of clinical expression correlates well with the immediate nutritional environment, age, culture, and eating habits of affected subjects. Here we review the biochemical, genetic, and molecular basis of human aldolase B deficiency in HFI, a disorder which responds to dietary therapy and in which the principal manifestations of disease are thus preventable.

Thermostable variants of Zymomonas mobilis alcohol dehydrogenase obtained using PCR-mediated random mutagenesis.

Using a random mutagenesis technique, the ferrous-ion-activated alcohol dehydrogenase of Zymomonas mobilis has been altered to produce more thermally stable variants. After three rounds of mutation, a variant over 10 degrees C more stable at pH 8, with essentially unaltered kinetic characteristics, was produced. However, the pH profile of thermostability of this variant was much altered compared with the wild-type, with a relatively small increase (4 degrees C) at pH 6. Sequencing of the variants indicated five amino acids changes which contributed to thermostability: F9S, M13I, K31R, F90L, and G250D. Four of these were contained in the final stable variant, and the changes were partially additive, with individual mutations causing between 2 and 3.5 degrees C stability increases (at pH 7.5). It is estimated that the most stable variant would have a half-life under physiological conditions at 70 degrees C of 15 min.

Alteration of substrate specificity of Zymomonas mobilis alcohol dehydrogenase-2 using in vitro random mutagenesis.

Random mutagenesis of the gene encoding Zymomonas mobilis alcohol dehydrogenase-2 has enabled isolation of variants of the enzyme that have substrate specificities different from that of the wild-type enzyme. After amino acids responsible for the changes were identified, directed mutation at these sites was also carried out. Variants that are active on butanol have been investigated in detail. Changes at residue 161 and other changes at residues 155 and 165 cause enhanced activity with longer-chain alcohols. The 165 change also induces a marked alcohol-activation phenomenon that is observed not only with ethanol, but also with a nonsubstrate alcohol, 2-propanol, and with low concentrations of Triton X-100. These alterations to the alcohol binding pocket mainly introduce larger, more hydrophobic residues, suggesting that it is not the size but the hydrophobicity of the pocket that affects the substrate specificity. Variants active with NADP were isolated, and, as with similar variants of the yeast enzyme, they were found to have an Asp residue replaced by a neutral amino acid. However, unlike the yeast examples in which the affinity was substantially reduced, the affinity for NAD+ in these variants was little changed, and the affinity for NADP+ was higher than that for NAD+. As this enzyme is naturally ferrous ion-activated, and inactive with zinc, attempts were made to find variants that had activity with zinc. One was found, but the screening method also isolated other variants with altered metal ion preferences due to a mutation affecting amino acid 330.

Neonatal screening for hereditary fructose intolerance: frequency of the most common mutant aldolase B allele (A149P) in the British population.

Hereditary fructose intolerance (HFI) causes severe and sometimes fatal metabolic disturbances in infants and children but responds to dietary treatment. To determine the practicability of screening newborn infants for HFI, we have investigated the frequency of the most common and widespread mutant allele of aldolase B, A149P, in the neonatal population. The polymerase chain reaction was used to amplify aldolase B exon 5 genomic sequences in DNA present in dried blood specimens preserved on Guthrie cards. The A149P mutation was identified by discriminatory hybridisation to allele specific oligonucleotides and confirmed independently by digestion with the restriction endonuclease BsaHI. Twenty-seven A149P heterozygotes were identified by the molecular analysis of aldolase B genes in blood samples obtained from a random cohort of 2050 subjects born in 1994 and 1995, 1.32 +/- 0.49% (95% confidence level). Although no A149P homozygotes were identified, the data allow the frequency of 1 in 23,000 homozygotes for this allele to be predicted. Our findings have implications for establishing an interventional mass screening programme to identify newborn infants with HFI in the UK.

Overexpression, purification, and generation of a thermostable variant of Zymomonas mobilis fructokinase.

The gene encoding fructokinase (EC 2.7.1.4) from Zymomonas mobilis has been expressed at high level in Escherichia coli by modifying the ribosome binding site using the polymerase chain reaction. A simple two-step purification from extracts of the recombinant cells results in highly purified enzyme suitable for use in fructose determination. Using the polymerase chain reaction in mutagenic conditions, a variant of fructokinase was isolated which was more thermostable than the wild type, taking the 30 min half-life from 70.1 to 72.4 degrees C. The purified thermostable variant had the same specific activity as the wild type. Sequencing of the variant indicated that only one amino acid was changed, with Ser 69 becoming Ala. Searches of the mutant libraries for variants that were (a) active with glucose or (b) had reduced inhibition by glucose were unsuccessful.

Polymerase chain reaction-based random mutagenesis: production and characterization of thermostable mutants of Zymomonas mobilis alcohol dehydrogenase-2.

The adhB gene encoding alcohol dehydrogenase-2 from Zymomonas mobilis has been subjected to random mutagenesis to obtain more thermostable variants of the enzyme. Random mutagenesis was accomplished using the polymerase chain reaction in mutagenic conditions. The optimum conditions involved restricting the concentration of one nucleotide to approximately one-tenth the normal amount. This introduced mutations at an average rate of 1 base in 600 in a 30-cycle PCR, sufficient to ensure that the majority of encoding DNA sequences in the mutant library have at least one base change from wild-type. Seven thermostable mutants were isolated from one library screening of 3000 colonies; two of these were selected for detailed study and purified using dye-ligand chromatography. Mutant TS-1 (F9S, V295A) was 3 degrees C more stable than the wild-type and had altered kinetic characteristics, with reduced affinity for ethanol and acetaldehyde and reduced ethanol oxidation Vmax. Mutant TS-2 (M13I, E19K, M192I) also had increased thermostability of 3 to 4 degrees C, but its kinetic characteristics were similar to that of the wild-type. Of the base changes found after sequencing a wide selection of mutants, 90% were transitions and 10% were transversions. Included were several T to C base changes which did not correspond with the nucleotide limitation used to create the mutant libraries.

Two promoters control the aroH gene of Escherichia coli.

The aroH gene from Escherichia coli encodes 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthase (Trp), one of three isoenzymes which catalyse the first committed step in the biosynthesis of aromatic amino acids and vitamins. S1 mapping and primer extension analysis of in vivo transcripts revealed the presence of two nonoverlapping promoters for aroH. The more distal of these has been described previously and is negatively regulated by the trp repressor. The second promoter is active under conditions of growth in rich medium, and may be involved in ensuring sufficient levels of precursors for the biosynthesis of aromatic vitamins under these growth conditions.