The novel P330L pathogenic variant of aromatic amino acid decarboxylase maps on the catalytic flexible loop underlying its crucial role

Aromatic amino acid decarboxylase (AADC) deficiency is a rare monogenic disease, often fatal in the first decade, causing severe intellectual disability, movement disorders and autonomic dysfunction. It is due to mutations in the gene coding for the AADC enzyme responsible for the synthesis of dopamine and serotonin. Using whole exome sequencing, we have identified a novel homozygous c.989C > T (p.Pro330Leu) variant of AADC causing AADC deficiency. Pro330 is part of an essential structural and functional element: the flexible catalytic loop suggested to cover the active site as a lid and properly position the catalytic residues. Our investigations provide evidence that Pro330 concurs in the achievement of an optimal catalytic competence. Through a combination of bioinformatic approaches, dynamic light scattering measurements, limited proteolysis experiments, spectroscopic and in solution analyses, we demonstrate that the substitution of Pro330 with Leu, although not determining gross conformational changes, results in an enzymatic species that is highly affected in catalysis with a decarboxylase catalytic efficiency decreased by 674- and 194-fold for the two aromatic substrates. This defect does not lead to active site structural disassembling, nor to the inability to bind the pyridoxal 5'-phosphate (PLP) cofactor. The molecular basis for the pathogenic effect of this variant is rather due to a mispositioning of the catalytically competent external aldimine intermediate, as corroborated by spectroscopic analyses and pH dependence of the kinetic parameters. Altogether, we determined the structural basis for the severity of the manifestation of AADC deficiency in this patient and discussed the rationale for a precision therapy

The Structure, Activity, and Function of the SETD3 Protein Histidine Methyltransferase

SETD3 has been recently identified as a long sought, actin specific histidine methyltransferase that catalyzes the Nτ-methylation reaction of histidine 73 (H73) residue in human actin or its equivalent in other metazoans. Its homologs are widespread among multicellular eukaryotes and expressed in most mammalian tissues. SETD3 consists of a catalytic SET domain responsible for transferring the methyl group from S-adenosyl-L-methionine (AdoMet) to a protein substrate and a RuBisCO LSMT domain that recognizes and binds the methyl-accepting protein(s). The enzyme was initially identified as a methyltransferase that catalyzes the modification of histone H3 at K4 and K36 residues, but later studies revealed that the only bona fide substrate of SETD3 is H73, in the actin protein. The methylation of actin at H73 contributes to maintaining cytoskeleton integrity, which remains the only well characterized biological effect of SETD3. However, the discovery of numerous novel methyltransferase interactors suggests that SETD3 may regulate various biological processes, including cell cycle and apoptosis, carcinogenesis, response to hypoxic conditions, and enterovirus pathogenesis. This review summarizes the current advances in research on the SETD3 protein, its biological importance, and role in various diseases

Molecular identification of carnosine N-methyltransferase as chicken histamine N-methyltransferase-like protein (hnmt-like).

Anserine (beta-alanyl-N(Pi)-methyl-L-histidine), a naturally occurring derivative of carnosine (beta-alanyl-L-histidine), is an abundant constituent of skeletal muscles and brain of many vertebrates. Although it has long been proposed to serve as a proton buffer, radicals scavenger and transglycating agent, its physiological function remains obscure. The formation of anserine is catalyzed by carnosine N-methyltransferase which exhibits unknown molecular identity. In the present investigation, we have purified carnosine N-methyltransferase from chicken pectoral muscle about 640-fold until three major polypeptides of about 23, 26 and 37 kDa coeluting with the enzyme were identified in the preparation. Mass spectrometry analysis of these polypeptides resulted in an identification of histamine N-methyltransferase-like (HNMT-like) protein as the only meaningful candidate. Analysis of GenBank database records indicated that the hnmt-like gene might be a paralogue of histamine N-methyltransferase gene, while comparison of their protein sequences suggested that HNMT-like protein might have acquired a new activity. Chicken HNMT-like protein was expressed in COS-7 cells, purified to homogeneity, and shown to catalyze the formation of anserine as confirmed by both chromatographic and mass spectrometry analysis. Both specificity and kinetic studies carried out on the native and recombinant enzyme were in agreement with published data. Particularly, several compounds structurally related to carnosine, including histamine and L-histidine, were tested as potential substrates for the enzyme, and carnosine was the only methyl group acceptor. The identification of the gene encoding carnosine N-methyltransferase might be beneficial for estimation of the biological functions of anserine

Vertebrate Acyl-CoA Synthetase Family Member 4 (ACSF4-U26) is a β-alanine activating enzyme homologous to bacterial nonribosomal peptide synthetase.

Mammalian ACSF4-U26 (Acyl-CoA Synthetase Family Member 4), a protein of unknown function, comprises a putative adenylation-domain (AMP-binding domain) similar to those of bacterial non-ribosomal peptide synthetases, a putative phosphopantetheine attachment site and a C-terminal PQQDH (pyrroloquinoline quinone dehydrogenase)-related domain. Orthologues comprising these three domains are present in many eukaryotes including plants. Remarkably, the adenylation domain of plant ACSF4-U26 shares more identity with Ebony, the insect enzyme that ligates β-alanine to several amines, than with vertebrate or insect ACSF4-U26 and prediction of its specificity suggests that it also serves to activate β-alanine. In the presence of ATP, purified mouse recombinant ACSF4-U26 progressively formed a covalent bond with radiolabelled β-alanine. The bond was alkali labile, suggesting a (thio)ester, and was not formed in a point-mutant devoid of the phosphopantetheine attachment site. Competition experiments with various amino acids indicated that the reaction was nearly specific for β-alanine, for which a KM of ≈ 5 µM was computed. The loaded enzyme was used to study the formation of a potential end-product. Among the twenty standard amino acids, only cysteine was able to cause unloading of the enzyme. This effect was mimicked by cysteamine and by dithiothreitol, and was unaffected by the absence of the PQQDH-related domain, suggesting that the β-alanine transfer onto thiols is catalyzed by the ACSF4-U26 adenylation domain, but is physiologically irrelevant. We conclude that ACSF4-U26 is a β-alanine activating enzyme, and hypothesize that it is involved in a rare intracellular reaction, possibly an infrequent post-translational or post-transcriptional modification. This article is protected by copyright. All rights reserved

Mass spectrum of a product formed by chicken HNMT-like protein.

<p>Homogenous recombinant chicken HNMT-like protein was incubated for 12 h with 3 mM carnosine in the absence or presence of 2 mM SAM. The produced methylated dipeptide was separated from the substrates and analyzed by mass spectrometry. Both mass spectra, covering the mass range <i>m/z</i> 50–600, and tandem mass spectra (Q-TOF) for anserine precursor ion (<i>m/z</i> 241) were acquired.</p

Amino acid sequence alignment of selected HNMT-like proteins with paralogue HNMT proteins.

<p>Sequences of turkey HNMT-like protein (GenBank Accession Number: XP_003207778.1), Zebra Finch HNMT-like protein (XP_002194298.1), the Green Anole HNMT-like protein (XP_003215153.1), the Nile Tilapia HNMT-like_1 and HNMT-like_2 proteins (XP_003445374.1 and XP_003445450.1, respectively), and the Sea Urchin HNMT-like_1 and HNMT-like_2 proteins (XP_786900.1 and XP_792696.1, respectively) were identified by Protein Blast searches with the use of chicken HNMT-like protein sequence (XP_001234740.1) and aligned with HNMT sequences of chicken (GenBank Accession Number: XP_422143.2), turkey (XP_003207782.1), Zebra Finch (XP_002194327.1) and the Green Anole (XP_003215142.1) using M-Coffee <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064805#pone.0064805-Wallace1" target="_blank">[27]</a>. Level of residues conservation is indicated by black (100%), dark grey (70% and more) and light gray (50% and more) background.</p

Purification of chicken carnosine N-methyltransferase.

<p>The enzyme was purified by chromatography on DEAE-Sepharose (A), Q-Sepharose (not shown), Superdex 200 (B), and Superdex 75 (C) as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064805#s2" target="_blank">Materials and Methods</a>” section. Fractions were tested for carnosine N-methyltransferase activity. Protein concentration was determined in the most active fractions with the Bradford assay. The indicated fractions eluted from the Superdex 75 column were analyzed by SDS-PAGE and the gel was stained with Coomassie Blue. The indicated bands were cut out of the gel, submitted to trypsin digestion and analyzed by tandem mass spectrometry.</p

Kinetic properties of chicken carnosine N-methyltransferase.

<p>Kinetic properties were determined with the use of homogenous recombinant chicken HNMT-like protein and chicken muscle carnosine N-methyltransferase purified by chromatography on DEAE-Sepharose, Q-Sepharose, Superdex 200 and Superdex 75.</p><p>Determinations for S-adenosyl-L-methionine (SAM) were performed with enzyme preparations that were incubated for 10–15 min in the presence of 20 mM carnosine and variable concentrations of (¹H+³H)SAM, while the measurements for carnosine were done in the presence of non-saturating 1 µM concentration of (¹H+³H)SAM. Values are the means of three separate experiments. The S.E. value is also given.</p

HPLC-HILIC analysis of a product formed by chicken HNMT-like protein.

<p>Chromatograms of standard mixture of carnosine and anserine (10 nmol) (A), of deproteinized reaction mixtures obtained from incubation of homogenous recombinant chicken HNMT-like protein for 12 h with 3 mM carnosine in the absence (B) or presence of 2 mM SAM (C) and following the supplementation of the former deproteinized reaction mixture with 20 nmol of anserine standard (D). The identity of all indicated compounds was confirmed by mass spectrometry. The sample processing and chromatographic conditions are described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064805#s2" target="_blank">Materials and Methods</a>”. AdoHcy, S-Adenosyl-L-homocysteine.</p

Amino acid sequence alignment of human HNMT with its chicken orthologue and chicken HNMT-like protein.

<p>Sequences were obtained with following GenBank accession numbers: human HNMT (NP_008826.1), chicken HNMT (XP_422143.2) and HNMT-like protein (XP_001234740.1). The chicken HNMT-like protein sequence has been confirmed by PCR amplification of the cDNA and sequencing. Percentage of amino acid identities with chicken HNMT is given in the upper right. Fully conserved residues are highlighted with a black background. Residues of human HNMT interacting with either SAM or histamine are marked by hashes or asterisks, respectively <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064805#pone.0064805-Horton1" target="_blank">[19]</a>. The peptides identified by mass spectrometry in the protein purified from chicken pectoral muscle are underlined in the chicken sequence.</p