Dataset Information

Molecular expression and characterization of erythroid-specific 5-aminolevulinate synthase gain-of-function mutations causing X-linked protoporphyria.

ABSTRACT: X-linked protoporphyria (XLP) (MIM 300752) is a recently recognized erythropoietic porphyria due to gain-of-function mutations in the erythroid-specific aminolevulinate synthase gene (ALAS2). Previously, two exon 11 small deletions, c.1699_1670?AT (?AT) and c.1706_1709?AGTG (?AGTG), that prematurely truncated or elongated the ALAS2 polypeptide, were reported to increase enzymatic activity 20- to 40-fold, causing the erythroid accumulation of protoporphyrins, cutaneous photosensitivity and liver disease. The mutant ?AT and ?AGTG ALAS2 enzymes, two novel mutations, c.1734?G (?G) and c.1642C>T (p.Q548X), and an engineered deletion c.1670-1671TC>GA p.F557X were expressed, and their purified enzymes were characterized. Wild-type and ?AGTG enzymes exhibited similar amounts of 54- and 52-kDa polypeptides on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), whereas the ?AT and p.F557X had only 52-kDa polypeptides. Compared to the purified wild-type enzyme, ?AT, ?AGTG and Q548X enzymes had increased specific activities that were only 1.8-, 3.1- and 1.6-fold, respectively. Interestingly, binding studies demonstrated that the increased activity Q548X enzyme did not bind to succinyl-CoA synthetase. The elongated ?G enzyme had wild-type specific activity, kinetics and thermostability; twice the wild-type purification yield (56 versus 25%); and was primarily a 54-kDa form, suggesting greater stability in vivo. On the basis of studies of mutant enzymes, the maximal gain-of function region spanned 57 amino acids between 533 and 580. Thus, these ALAS2 gain-of-function mutations increased the specific activity (?AT, ?AGTG and p.Q548X) or stability (?G) of the enzyme, thereby leading to the increased erythroid protoporphyrin accumulation causing XLP.

SUBMITTER: Bishop DF

PROVIDER: S-EPMC3592931 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature

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Molecular expression and characterization of erythroid-specific 5-aminolevulinate synthase gain-of-function mutations causing X-linked protoporphyria.

Bishop David F DF Tchaikovskii Vassili V Nazarenko Irina I Desnick Robert J RJ

Molecular medicine (Cambridge, Mass.) 20130305

X-linked protoporphyria (XLP) (MIM 300752) is a recently recognized erythropoietic porphyria due to gain-of-function mutations in the erythroid-specific aminolevulinate synthase gene (ALAS2). Previously, two exon 11 small deletions, c.1699_1670ΔAT (ΔAT) and c.1706_1709ΔAGTG (ΔAGTG), that prematurely truncated or elongated the ALAS2 polypeptide, were reported to increase enzymatic activity 20- to 40-fold, causing the erythroid accumulation of protoporphyrins, cutaneous photosensitivity and liver ...[more]

PMID: 23348515

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Project description:5-Aminolevulinate synthase is the first enzyme of the heme biosynthetic pathway in non-plant eukaryotes and some prokaryotes. The enzyme functions as a homodimer and requires pyridoxal 5'-phosphate as a cofactor. Although the roles of defined amino acids in the active site and catalytic mechanism have been recently explored using site-directed mutagenesis, much less is known about the role of the 5-aminolevulinate synthase polypeptide chain arrangement in folding, structure, and ultimately, function. To assess the importance of the continuity of the polypeptide chain, circularly permuted 5-aminolevulinate synthase variants were constructed through either rational design or screening of an engineered random library. One percent of the random library clones were active, and a total of 21 active variants had sequences different from that of the wild type 5-aminolevulinate synthase. Out of these 21 variants, 9 displayed unique circular permutations of the 5-aminolevulinate synthase polypeptide chain. The new termini of the active variants disrupted secondary structure elements and loop regions and fell in 100 amino acid regions from each terminus. This indicates that the natural continuity of the 5-aminolevulinate synthase polypeptide chain and the sequential arrangement of the secondary structure elements are not requirements for proper folding, binding of the cofactor, or assembly of the two subunits. Furthermore, the order of two identified functional elements (i.e. the catalytic and the glycine-binding domains) is apparently irrelevant for proper functioning of the enzyme. Although the wild type 5-aminolevulinate synthase and the circularly permuted variants appear to have similar, predicted overall tertiary structures, they exhibit differences in the arrangement of the secondary structure elements and in the cofactor-binding site environment. Taken together, the data lead us to propose that the 5-aminolevulinate synthase overall structure can be reached through multiple or alternative folding pathways.

Dataset Information

Molecular expression and characterization of erythroid-specific 5-aminolevulinate synthase gain-of-function mutations causing X-linked protoporphyria.

Publications

Molecular expression and characterization of erythroid-specific 5-aminolevulinate synthase gain-of-function mutations causing X-linked protoporphyria.

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