An Arabidopsis Oxalyl-CoA Decarboxylase, AtOXC, Is Important for Oxalate Catabolism in Plants

Considering the widespread occurrence of oxalate in nature and its broad impact on a host of organisms, it is surprising that so little is known about the turnover of this important acid. In plants, oxalate oxidase is the most well-studied enzyme capable of degrading oxalate, but not all plants possess this activity. Recently, acyl-activating enzyme 3 (AAE3), encoding an oxalyl-CoA synthetase, was identified in Arabidopsis. This enzyme has been proposed to catalyze the first step in an alternative pathway of oxalate degradation. Since this initial discovery, this enzyme and proposed pathway have been found to be important to other plants and yeast as well. In this study, we identify, in Arabidopsis, an oxalyl-CoA decarboxylase (AtOXC) that is capable of catalyzing the second step in this proposed pathway of oxalate catabolism. This enzyme breaks down oxalyl-CoA, the product of AtAAE3, into formyl-CoA and CO2. AtOXC:GFP localization suggested that this enzyme functions within the cytosol of the cell. An Atoxc knock-down mutant showed a reduction in the ability to degrade oxalate into CO2. This reduction in AtOXC activity resulted in an increase in the accumulation of oxalate and the enzyme substrate, oxalyl-CoA. Size exclusion studies suggest that the enzyme functions as a dimer. Computer modeling of the AtOXC enzyme structure identified amino acids of predicted importance in co-factor binding and catalysis. Overall, these results suggest that AtOXC catalyzes the second step in this alternative pathway of oxalate catabolism.

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Rapid Organocatalytic Chirality Analysis of Amines, Amino Acids, Alcohols, Amino Alcohols and Diols with Achiral Iso(thio)cyanate Probes

Chemical Science ◽

10.1039/d1sc02061g ◽

2021 ◽

Author(s):

Eryn Nelson ◽

Jeffrey S. S. K. Formen ◽

Christian Wolf

Keyword(s):

Amino Acids ◽

Absolute Configuration ◽

Rapid Determination ◽

Amino Alcohols ◽

Enantioselective Synthesis ◽

Widespread Occurrence ◽

Chiral Compounds ◽

New Methods ◽

The Absolute

The widespread occurrence and significance of chiral compounds does not only require new methods for their enantioselective synthesis but also efficient tools that allow rapid determination of the absolute configuration,...

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Oxalic acid degradation by a novel fungal oxalate oxidase from Abortiporus biennis

Acta Biochimica Polonica ◽

10.18388/abp.2016_1282 ◽

2016 ◽

Vol 63 (3) ◽

Cited By ~ 3

Author(s):

Marcin Grąz ◽

Kamila Rachwał ◽

Radosław Zan ◽

Anna Jarosz-Wilkołazka

Keyword(s):

Oxalic Acid ◽

Ph Value ◽

Enzyme Reaction ◽

Oxalate Oxidase ◽

Ion Exchange Chromatography ◽

Exchange Chromatography ◽

Size Exclusion ◽

Optimum Temperature ◽

Intracellular Enzyme ◽

Exclusion Chromatography

Oxalate oxidase was identified in mycelial extracts of a basidiomycete Abortiporus biennis strain. Intracellular enzyme activity was detected only after prior lowering of the pH value of the fungal cultures by using oxalic or hydrochloric acids. This enzyme was purified using size exclusion chromatography (Sephadex G-25) and ion-exchange chromatography (DEAE-Sepharose). This enzyme exhibited optimum activity at pH 2 when incubated at 40°C, and the optimum temperature was established at 60°C. Among the tested organic acids, this enzyme exhibited specificity only towards oxalic acid. Molecular mass was calculated as 58 kDa. The values of Km for oxalate and Vmax for the enzyme reaction were 0.015 M and 30 mmol min-1, respectively.

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Structural characterization, tissue distribution, and functional expression of murine aminoacylase III

AJP Cell Physiology ◽

10.1152/ajpcell.00192.2003 ◽

2004 ◽

Vol 286 (4) ◽

pp. C848-C856 ◽

Cited By ~ 58

Author(s):

Alexander Pushkin ◽

Gerardo Carpenito ◽

Natalia Abuladze ◽

Debra Newman ◽

Vladimir Tsuprun ◽

...

Keyword(s):

Amino Acids ◽

Molecular Mass ◽

Rotational Symmetry ◽

Aromatic Amino Acids ◽

Functional Expression ◽

Proximal Tubules ◽

Size Exclusion ◽

Northern Analysis ◽

Mercapturic Acids

Many xenobiotics are detoxified through the mercapturate metabolic pathway. The final product of the pathway, mercapturic acids ( N-acetylcysteine S-conjugates), are secreted predominantly by renal proximal tubules. Mercapturic acids may undergo a transformation mediated by aminoacylases and cysteine S-conjugate β-lyases that leads to nephrotoxic reactive thiol formation. The deacetylation of cysteine S-conjugates of N-acyl aromatic amino acids is thought to be mediated by an aminoacylase whose molecular identity has not been determined. In the present study, we cloned aminoacylase III, which likely mediates this process in vivo, and characterized its function and structure. The enzyme consists of 318 amino acids and has a molecular mass (determined by SDS-PAGE) of ∼35 kDa. Under nondenaturing conditions, the molecular mass of the enzyme is ∼140 kDa as determined by size-exclusion chromatography, which suggests that it is a tetramer. In agreement with this hypothesis, transmission electron microscopy and image analysis of aminoacylase III showed that the monomers of the enzyme are arranged with a fourfold rotational symmetry. Northern analysis demonstrated an ∼1.4-kb transcript that was expressed predominantly in kidney and showed less expression in liver, heart, small intestine, brain, lung, testis, and stomach. In kidney, aminoacylase III was immunolocalized predominantly to the apical domain of S1 proximal tubules and the cytoplasm of S2 and S3 proximal tubules. The data suggest that in kidney proximal tubules, aminoacylase III plays an important role in deacetylating mercapturic acids. The predominant cytoplasmic localization of aminoacylase III may explain the greater sensitivity of the proximal straight tubule to the nephrotoxicity of mercapturic acids.

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A Dimerization Site at SCR-17/18 in Factor H Clarifies a New Mechanism for Complement Regulatory Control

Frontiers in Immunology ◽

10.3389/fimmu.2020.601895 ◽

2021 ◽

Vol 11 ◽

Author(s):

Orla M. Dunne ◽

Xin Gao ◽

Ruodan Nan ◽

Jayesh Gor ◽

Penelope J. Adamson ◽

...

Keyword(s):

Host Cell ◽

Analytical Ultracentrifugation ◽

Dissociation Constants ◽

Alternative Pathway ◽

Factor H ◽

Regulatory Control ◽

Size Exclusion ◽

Complement Factor ◽

Dimer Formation ◽

Self Association

Complement Factor H (CFH), with 20 short complement regulator (SCR) domains, regulates the alternative pathway of complement in part through the interaction of its C-terminal SCR-19 and SCR-20 domains with host cell-bound C3b and anionic oligosaccharides. In solution, CFH forms small amounts of oligomers, with one of its self-association sites being in the SCR-16/20 domains. In order to correlate CFH function with dimer formation and the occurrence of rare disease-associated variants in SCR-16/20, we identified the dimerization site in SCR-16/20. For this, we expressed, in Pichia pastoris, the five domains in SCR-16/20 and six fragments of this with one-three domains (SCR-19/20, SCR-18/20, SCR-17/18, SCR-16/18, SCR-17 and SCR-18). Size-exclusion chromatography suggested that SCR dimer formation occurred in several fragments. Dimer formation was clarified using analytical ultracentrifugation, where quantitative c(s) size distribution analyses showed that SCR-19/20 was monomeric, SCR-18/20 was slightly dimeric, SCR-16/20, SCR-16/18 and SCR-18 showed more dimer formation, and SCR-17 and SCR-17/18 were primarily dimeric with dissociation constants of ~5 µM. The combination of these results located the SCR-16/20 dimerization site at SCR-17 and SCR-18. X-ray solution scattering experiments and molecular modelling fits confirmed the dimer site to be at SCR-17/18, this dimer being a side-by-side association of the two domains. We propose that the self-association of CFH at SCR-17/18 enables higher concentrations of CFH to be achieved when SCR-19/20 are bound to host cell surfaces in order to protect these better during inflammation. Dimer formation at SCR-17/18 clarified the association of genetic variants throughout SCR-16/20 with renal disease.

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An exploratory study of pre-extrusion flavouring: investigation with vitamins, amino acids, essential oils, natural aromas and seasonings

Acta Scientiarum Technology ◽

10.4025/actascitechnol.v43i1.49956 ◽

2020 ◽

Vol 43 ◽

pp. e49956

Author(s):

Leonardo Heleno Milk ◽

Talita Maira Goss Milani ◽

Ana Carolina Conti-Silva

Keyword(s):

Amino Acids ◽

Essential Oils ◽

Nutritive Value ◽

Negative Impact ◽

Positive Impact ◽

Expansion Ratio ◽

Second Step ◽

Corn Snacks ◽

The Impact ◽

Lipid Vehicle

Expanded corn snacks are obtained through thermoplastic extrusion and then a lipid vehicle (oil or hydrogenated vegetable fat) is sprinkled on the product, giving the desired flavour. However, this second step called post-extrusion flavouring increases the lipid content and the caloric value of the snack, reasons why these products are highly criticized. Thus, the study of pre-extrusion flavourings is an interesting way of finding corn snacks with better nutritive value. Therefore, we aimed to explore pre-extrusion flavouring using different substances, investigating the odour and flavour on the extrudates and the impact of such substances on their expansion ratio, density, and colour. To do this, vitamins, amino acids, essential oils, natural aromas and seasonings were added to corn grits and extruded. The addition of different substances, especially the essential oils, natural aromas and seasonings, produced odour and flavour in the extrudates, as well as having little negative impact on their physical properties of the extrudates, no impact or even a small positive impact. Therefore, this study provides a range of possibilities to be explored, using pre-extrusion flavouring as a way of reducing the use of lipids and improving the nutritive value of expanded snacks.

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Crystallization and preliminary X-ray diffraction studies of the C-terminal domain ofChlamydia trachomatisCdsD

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x14019712 ◽

2014 ◽

Vol 70 (10) ◽

pp. 1431-1433 ◽

Cited By ~ 1

Author(s):

Gitte Meriläinen ◽

Rik K. Wierenga

Keyword(s):

Amino Acids ◽

Type Iii Secretion ◽

Type Iii Secretion System ◽

Secretion System ◽

Size Exclusion ◽

Diffusion Method ◽

Data Set ◽

Cell Parameters ◽

Type Iii ◽

Terminal Domain

The inner membrane ring of the bacterial type III secretion system (TTSS) is composed of two proteins. InChlamydia trachomatisthis ring is formed by CdsD (gene nameCT_664) and CdsJ (gene nameCTA_0609). CdsD consists of 829 amino acids. The last 400 amino acids at its C-terminal end relate it to the type III secretion system YscD/HrpQ protein family. The C-terminal domain, consisting of amino acids 558–771, ofC. trachomatisCdsD was overexpressed inEscherichia coliand purified using immobilized metal-affinity chromatography (IMAC) and size-exclusion chromatography. The protein was crystallized using the vapour-diffusion method. A data set was collected to 2.26 Å resolution. The crystals have the symmetry of space groupC2, with unit-cell parametersa= 106.60,b= 23.91,c= 118.65 Å, β = 104.95°. According to the data analysis there is expected to be one molecule in the asymmetric unit, with a Matthews coefficient of 3.0 Å3 Da−1.

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The NHR2 Oligomerization Domain of AML1/ETO as a Novel Therapeutic Target Structure in t(8;21) Positive Leukemias.

Blood ◽

10.1182/blood.v110.11.210.210 ◽

2007 ◽

Vol 110 (11) ◽

pp. 210-210

Author(s):

Christian Wichmann ◽

Yvonne Becker ◽

Manuel Grez

Keyword(s):

Cell Cycle ◽

Amino Acids ◽

Binding Capacity ◽

Growth Arrest ◽

Laser Scanning Microscopy ◽

Size Exclusion ◽

Myeloid Differentiation ◽

Protein Transduction ◽

Dependent Manner ◽

Cellular Expression

Abstract About 12% of all de novo acute myeloid leukemias are characterized by the translocation t(8;21) which generates the oncogenic fusion protein AML1/ETO. AML1/ETO is found in high molecular weight complexes (HMWC) which are crucial for the block in myeloid differentiation of AML1/ETO transformed cells. Essential for HMWC formation is the alpha-helical nervy homology region 2 (NHR2) within ETO which serves as an interacting surface for oligomerization as well as association with members of the ETO protein family. Based on the observation that the integrity of the NHR2 domain is essential for transcriptional repressor activity of ETO, we designed a peptide (NC128) aimed to selectively interfere with AML1/ETO oligomerization. Confocal laser scanning microscopy revealed colocalization of NC128 with AML1/ETO in the nucleus. In protein-protein interaction studies we could demonstrate that the NHR2 domain in NC128 was necessary and sufficient for proper binding to AML1/ETO. Expression of NC128 in AML1/ETO positive cells led to inhibition of AML1/ETO HMWC formation as demonstrated by size-exclusion chromatography. NC128 expression in the AML1/ETO growth dependent Kasumi-1 cells restores transcription of AML1/ETO target genes. Among others, the expression of PU.1, a master regulator of myeloid differentiation, was significantly upregulated in the presence of NC128. In Kasumi-1 cells as well as in U937-AML1/ETO cells expression of NC128 almost completely overcomes the block of cytokine mediated differentiation induced by AML1/ETO. In the presence of NC128, Kasumi-1 cells lose their progenitor cell characteristics and upregulate marker of early monocytic differentiation. Likewise, NC128 expressing Kasumi-1 and SKNO-1 cells are arrested in cell cycle progression with a peak in the G1 phase of the cell cycle. The binding affinity of NHR2 peptide mutants to ETO directly correlates with the intensity of growth inhibition. A derivative of NC128 which retained all NHR2 amino acids (N89) maintained full binding capacity to ETO as well as antiproliferative effects, whereas a mutant of N89 lacking 7 C-terminal amino acids (N82) significantly lost binding capacity and its antiproliferative effect. Next a codon optimized expression construct was developed in order to increase the cellular expression levels of N89. Compared to N89, expression of this construct enhances growth arrest suggesting that the NHR2 peptides act in a dose dependent manner. To investigate the influence of NC128 on primary cells, human CD34 progenitor cells were immortalized with AML1/ETO. Expression of NC128 in these cells resulted in growth arrest, loss of CD34 expression and an increased rate of apoptosis. In order to directly deliver the peptide to AML1/ETO expressing cells, the NHR2 domain was fused to the HIV-1 Tat protein transduction domain. The bacterial expressed peptides were purified by affinity chromatography and were proven to be stable in serum-containing medium for several hours. Upon protein transduction into mammalian cells, recombinant NHR2 peptides could be detected in cellular lysates. Furthermore, we already can show by co-immunoprecipitation experiments that the transducible peptides are able to specifically interact with ETO protein. Our results propose that selective interference with NHR2-mediated oligomerization could provide a promising strategy for the inhibition of the oncogenic properties of AML1/ETO. Based on our results, we are aiming to develop screening strategies for both peptides and small molecule substances to interfere with NHR2 mediated AML1/ETO complex formation.

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Amino acid sequence of HSP-1, a major protein of stallion seminal plasma: effect of glycosylation on its heparin- and gelatin-binding capabilities

Biochemical Journal ◽

10.1042/bj3100615 ◽

1995 ◽

Vol 310 (2) ◽

pp. 615-622 ◽

Cited By ~ 70

Author(s):

J J Calvete ◽

K Mann ◽

W Schäfer ◽

L Sanz ◽

M Reinert ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Amino Acid Sequence ◽

Seminal Plasma ◽

Binding Activity ◽

Size Exclusion ◽

Major Protein ◽

Heparin Binding ◽

Aggregation State ◽

Consensus Pattern

We report the complete amino acid sequence of HSP-1, a major protein isolated from stallion seminal plasma or acid extracts of ejaculated spermatozoa. The protein consists of 121 amino acids organized in two types of homologous repeats arranged in the pattern AA‘BB’. Each of the 13-15-residue A-type repeats contains two O-linked oligosaccharide chains. The B-type repeats span 44-47 amino acids each, are not glycosylated, and have the consensus pattern of the gelatin-binding fibronectin type-II module. This domain also occurs in the major bovine seminal plasma heparin-binding proteins PDC-109 (BSP-A1/A2) and BSP-A3. However, unlike the bovine proteins which bind quantitatively to a heparin-Sepharose column, stallion HSP-1 was recovered in both the flow-through and the heparin-bound fractions. Structural analysis showed that the two HSP-1 forms contain identical polypeptide chains which are differently glycosylated. Moreover, size-exclusion chromatography showed that heparin-bound HSP-1 associates with HSP-2, another major seminal plasma protein, into a 90 kDa product, whereas the non-heparin-bound glycoform of HSP-1 is eluted as a monomeric (14 kDa) protein. This suggests that glycosylation may have an indirect effect on the heparin-binding ability of HSP-1 through modulation of its aggregation state. On the other hand, both glycoforms of HSP-1 displayed gelatin-binding activity, indicating that the molecular determinants for binding heparin and gelatin are different.

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The Linker Domain of SNAP25 Acts as a Flexible Molecular Spacer to Ensure Efficient S-Acylation

10.1101/2020.01.21.914333 ◽

2020 ◽

Author(s):

Christine Salaun ◽

Jennifer Greaves ◽

Nicholas C.O. Tomkinson ◽

Luke H. Chamberlain

Keyword(s):

Amino Acids ◽

Binding Motif ◽

Linear Motif ◽

Linker Region ◽

Rich Domain ◽

Enzyme Substrate ◽

Efficient Coupling ◽

Affinity Interaction ◽

Linker Domain ◽

Cysteine Rich Domain

ABSTRACTS-Acylation of the SNARE protein SNAP25 is mediated by a subset of Golgi zDHHC enzymes, in particular zDHHC17. The ankyrin repeat (ANK) domain of this enzyme interacts with a short linear motif known as the zDHHC ANK binding motif (zDABM) in SNAP25 (112-VVASQP-117), which is downstream of the S-acylated cysteine-rich domain (85-CGLCVCPC-92). In this study, we have investigated the importance of the flexible linker (amino acids 93-111; referred to as the “mini-linker” region) that separates the zDABM and S-acylated cysteines. Shortening the mini-linker had no effect of zDHHC17 interaction but blocked S-acylation. Insertion of additional flexible glycine-serine repeats had no effect on S-acylation, whereas extended and rigid alanine-proline repeats perturbed this process. Indeed, a SNAP25 mutant in which the mini-linker region was substituted with a flexible glycine-serine linker of the same length underwent efficient S-acylation. Furthermore, this mutant displayed the same intracellular localisation as wild-type SNAP25, showing that the sequence of the mini-linker is not important in this context. By using the results of previous peptide array experiments, we generated a SNAP25 mutant predicted to have a higher affinity zDABM, and this mutant showed enhanced interaction with zDHHC17 in cells. Interestingly, this mutant was S-acylated with reduced efficiency, implying that a lower affinity interaction of the SNAP25 zDABM with zDHHC17 is optimal for S-acylation efficiency. Overall, the results of this study show that amino acids 93-111 in SNAP25 act as a flexible molecular spacer to ensure efficient coupling of enzyme-substrate interaction and S-acylation.

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BODIPY- and Porphyrin-Based Sensors for Recognition of Amino Acids and Their Derivatives

Molecules ◽

10.3390/molecules25194523 ◽

2020 ◽

Vol 25 (19) ◽

pp. 4523 ◽

Cited By ~ 1

Author(s):

Marco Farinone ◽

Karolina Urbańska ◽

Miłosz Pawlicki

Keyword(s):

Amino Acids ◽

Molecular Recognition ◽

Biological Systems ◽

Direct Interaction ◽

Covalent Bonding ◽

Molecular Probes ◽

Chiral Discrimination ◽

Living Matter ◽

Diverse Range ◽

Widespread Occurrence

Molecular recognition is a specific non-covalent and frequently reversible interaction between two or more systems based on synthetically predefined character of the receptor. This phenomenon has been extensively studied over past few decades, being of particular interest to researchers due to its widespread occurrence in biological systems. In fact, a straightforward inspiration by biological systems present in living matter and based on, e.g., hydrogen bonding is easily noticeable in construction of molecular probes. A separate aspect also incorporated into the molecular recognition relies on the direct interaction between host and guest with a covalent bonding. To date, various artificial systems exhibiting molecular recognition and based on both types of interactions have been reported. Owing to their rich optoelectronic properties, chromophores constitute a broad and powerful class of receptors for a diverse range of substrates. This review focuses on BODIPY and porphyrin chromophores as probes for molecular recognition and chiral discrimination of amino acids and their derivatives.

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