Murine erythroid 5-aminolevulinate synthase: Truncation of a disordered N-terminal extension is not detrimental for catalysis

Stojanovski, Bosko M.; Breydo, Leonid; Uversky, Vladimir N.; Ferreira, Glória C.

doi:10.1016/j.bbapap.2016.02.002

Cited by 9 publications

(9 citation statements)

References 49 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

Section: N-terminal Peptide Deletion Influences Immunological and Strmentioning

confidence: 98%

“…The presence of such region turns cleavage sites more available, facilitating the activity of enzymes. 8 The identification of the generated proteolytic peptides revealed that the shortening of Blo t 5 did not alter the peptide cluster formation compared with the full-length molecule ( Figure S3). We confirmed that rBlo t 5 has no IgE epitopes at the disordered N-terminal motif (Figure 2A), as was reported by other research groups, who located the key amino acids for IgE binding at other positions of the allergen.…”

Section: N-terminal Peptide Deletion Influences Immunological and Strmentioning

confidence: 98%

See 1 more Smart Citation

N‐terminal peptide deletion influences immunological and structural features of Blo t 5

Silva

Huber

Alcantara-Neves

et al. 2020

Allergy

View full text Add to dashboard Cite

LETTERS TO THE EDITOR in infrequent phenotypes such as isolated angioedema by SRs and multiple SRs, which may be misdiagnosed as CRs. This procedure is useful not only for reducing the number of patients who are unnecessarily avoiding NSAIDs but also for extending their therapeutic possibilities. Although such accurate diagnosis requires facilities and trained personal in this field, it will improve the allergological workup and, consequently, patients' management.

show abstract

Section: N-terminal Peptide Deletion Influences Immunological and Strmentioning

confidence: 98%

Section: N-terminal Peptide Deletion Influences Immunological and Strmentioning

confidence: 98%

N‐terminal peptide deletion influences immunological and structural features of Blo t 5

Silva

Huber

Alcantara-Neves

et al. 2020

Allergy

View full text Add to dashboard Cite

show abstract

“…The shortened half-lives for thermal denaturation of the ALAS2 truncation mutations was consistent with an increase in instability and flexibility of the protein that could result in the observed increased in enzymatic activity due to increased rates of ALA release. Stojanovski, et al, has also recently suggested that truncation mutations that change the ALAS2 tertiary structure from a closed to open conformation may increase activity by releasing product more rapidly and may decrease thermostability by reducing the rigidity of the structure (Stojanovski et al 2016).…”

Section: Discussionmentioning

confidence: 99%

Molecular expression, characterization and mechanism of ALAS2 gain-of-function mutants

Tchaikovskii

Desnick

Bishop

2019

Mol Med

View full text Add to dashboard Cite

BackgroundX-linked protoporphyria (XLP) (MIM 300752) is an erythropoietic porphyria due to gain-of-function mutations in the last exon (Ducamp et al., Hum Mol Genet 22:1280-88, 2013) of the erythroid-specific aminolevulinate synthase gene (ALAS2). Five ALAS2 exon 11 variants identified by the NHBLI Exome sequencing project (p.R559H, p.E565D, p.R572C, p.S573F and p.Y586F) were expressed, purified and characterized in order to assess their possible contribution to XLP. To further characterize the XLP gain-of-function region, five novel ALAS2 truncation mutations (p.P561X, p.V562X, p.H563X, p.E569X and p.F575X) were also expressed and studied.MethodsSite-directed mutagenesis was used to generate ALAS2 mutant clones and all were prokaryotically expressed, purified to near homogeneity and characterized by protein and enzyme kinetic assays. Standard deviations were calculated for 3 or more assay replicates.ResultsThe five ALAS2 single nucleotide variants had from 1.3- to 1.9-fold increases in succinyl-CoA Vmax and 2- to 3-fold increases in thermostability suggesting that most could be gain-of-function modifiers of porphyria instead of causes. One SNP (p.R559H) had markedly low purification yield indicating enzyme instability as the likely cause for XLSA in an elderly patient with x-linked sideroblastic anemia. The five novel ALAS2 truncation mutations had increased Vmax values for both succinyl-CoA and glycine substrates (1.4 to 5.6-fold over wild-type), while the Kms for both substrates were only modestly changed. Of interest, the thermostabilities of the truncated ALAS2 mutants were significantly lower than wild-type, with an inverse relationship to Vmax fold-increase.ConclusionsPatients with porphyrias should always be assessed for the presence of the ALAS2 gain-of-function modifier variants identified here. A key region of the ALAS2 carboxyterminal region is identified by the truncation mutations studied here and the correlation of increased thermolability with activity suggests that increased molecular flexibility/active site openness is the mechanism of enhanced function of mutations in this region providing further insights into the role of the carboxyl-terminal region of ALAS2 in the regulation of erythroid heme synthesis.Electronic supplementary materialThe online version of this article (10.1186/s10020-019-0070-9) contains supplementary material, which is available to authorized users.

show abstract

“…The N-terminal 10 residues of Thermotoga maritima CE7 acetyl esterase played a role in substrate specificity and was important for thermal stability [24]. The N-terminal 36 residues of the murine 5-aminolevulinate synthase truncated product resulted in a lower thermal stability and increased k c a t value, and its N-terminus was involved in a cell-specific regulatory mechanism [25]. We proposed that the Nterminal 10 residues of CvPAH play a role in substrate binding or regulation given that the truncation decreased substrate affinity.…”

Section: Discussionmentioning

confidence: 99%

Catalytic Ability Improvement of Phenylalanine Hydroxylase from Chromobacterium violaceum by N-Terminal Truncation and Proline Introduction

Liu¹,

Cheng²,

Ye³

et al. 2019

Journal of Microbiology and Biotechnology

View full text Add to dashboard Cite

Phenylalanine hydroxylase from Chromobacterium violaceum (CvPAH) is a monomeric enzyme that converts phenylalanine to tyrosine. It shares high amino acid identity and similar structure with a subunit of human phenylalanine hydroxylase that is a tetramer, resulting in the latent application in medications. In this study, semirational design was applied to CvPAH to improve the catalytic ability based on molecular dynamics simulation analyses. Four Nterminal truncated variants and one single point variant were constructed and characterized. The D267P variant showed a 2.1-fold increased thermal stability compared to the wild type, but lower specific activity was noted compared with the wild type. The specific activity of all truncated variants was a greater than 25% increase compared to the wild type, and these variants showed similar or slightly decreased thermostability with the exception of the N-Δ9 variant. Notably, the N-Δ9 variant exhibited a 1.2-fold increased specific activity, a 1.3-fold increased thermostability and considerably increased catalytic activity under the neutral environment compared with the wild type. These properties of the N-Δ9 variant could advance medical and pharmaceutical applications of CvPAH. Our findings indicate that the N-terminus might modulate substrate binding, and are directives for further modification and functional research of PAH and other enzymes.

show abstract

Murine erythroid 5-aminolevulinate synthase: Truncation of a disordered N-terminal extension is not detrimental for catalysis

Cited by 9 publications

References 49 publications

N‐terminal peptide deletion influences immunological and structural features of Blo t 5

N‐terminal peptide deletion influences immunological and structural features of Blo t 5

Molecular expression, characterization and mechanism of ALAS2 gain-of-function mutants

Catalytic Ability Improvement of Phenylalanine Hydroxylase from Chromobacterium violaceum by N-Terminal Truncation and Proline Introduction

Contact Info

Product

Resources

About