l-Amino acid oxidase of the fungus Hebeloma cylindrosporum displays substrate preference towards glutamate

Nuutinen, Jaro T.; Marttinen, Eeva; Soliymani, Rabah; Hildén, Kristiina; Timonen, Sari

doi:10.1099/mic.0.054486-0

Cited by 30 publications

(20 citation statements)

References 57 publications

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“…The L-amino acid oxidases (LAAOs, EC 1.4.3.2) are flavoenzymes found in such diverse organisms as bacteria, fungi, algae, fish, snails as well as venoms of snakes from the families Viperidae, Crotalidae and Elapidae [1-6]. …”

Section: Introductionmentioning

confidence: 99%

Snake venom L-amino acid oxidases: an overview on their antitumor effects

Costa

Burin

Menaldo

et al. 2014

J Venom Anim Toxins incl Trop Dis

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The L-amino acid oxidases (LAAOs) constitute a major component of snake venoms and have been widely studied due to their widespread presence and various effects, such as apoptosis induction, cytotoxicity, induction and/or inhibition of platelet aggregation, hemorrhage, hemolysis, edema, as well as antimicrobial, antiparasitic and anti-HIV activities. The isolated and characterized snake venom LAAOs have become important research targets due to their potential biotechnological applications in pursuit for new drugs of interest in the scientific and medical fields. The current study discusses the antitumor effects of snake venom LAAOs described in the literature to date, highlighting the mechanisms of apoptosis induction proposed for this class of proteins.

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Section: Introductionmentioning

confidence: 99%

Snake venom L-amino acid oxidases: an overview on their antitumor effects

Costa

Burin

Menaldo

et al. 2014

J Venom Anim Toxins incl Trop Dis

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“…Therefore, we refer to the FAD-dependent type as LAAO. LAAOs with broad substrate selectivity, or promiscuous LAAOs, have been discovered in a diverse range of species, including bacteria (9), fungi (10), and vipers (11).…”

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confidence: 99%

Following the Evolutionary Track of a Highly Specific l -Arginine Oxidase by Reconstruction and Biochemical Analysis of Ancestral and Native Enzymes

Nakano

Niwa

Asano

et al. 2019

Appl Environ Microbiol

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Following the evolutionary track of enzymes can help elucidate how enzymes attain their characteristic functions, such as thermal adaptation and substrate selectivity, during the evolutionary process. Ancestral sequence reconstruction (ASR) is effective for following evolutionary processes if sufficient sequence data are available. Selecting sequences from the data to generate a curated sequence library is necessary for the successful design of artificial proteins by ASR. In this study, we tried to follow the evolutionary track of l-arginine oxidase (AROD), a flavin adenine dinucleotide (FAD)-dependent amino acid oxidase (LAAO) that exhibits high specificity for l-arginine. The library was generated by selecting sequences in which the 15th, 50th, 332nd, and 580th residues are Gly, Ser, Trp, and Thr, respectively. We excluded sequences that are either extremely short or long and those with a low degree of sequence identity. Three ancestral ARODs (AncARODn0, AncARODn1, and AncARODn2) were designed using the library. Subsequently, we expressed the ancestral ARODs as well as native Oceanobacter kriegii AROD (OkAROD) in bacteria. AncARODn0 is phylogenetically most remote from OkAROD, whereas AncARODn2 is most similar to OkAROD. Thermal stability was gradually increased by extending AROD sequences back to the progenitor, while the temperature at which the residual activity is half of the maximum measured activity (T1/2) of AncARODn0 was >20°C higher than that of OkAROD. Remarkably, only AncARODn0 exhibited broad substrate selectivity similar to that of conventional promiscuous LAAO. Taken together, our findings led us to infer that AROD may have evolved from a highly thermostable and promiscuous LAAO. IMPORTANCE In this study, we attempted to infer the molecular evolution of a recently isolated FAD-dependent l-arginine oxidase (AROD) that oxidizes l-arginine to 2-ketoarginine. Utilizing 10 candidate AROD sequences, we obtained a total of three ancestral ARODs. In addition, one native AROD was obtained by cloning one of the candidate ARODs. The candidate sequences were selected utilizing a curation method defined in this study. All the ARODs were successfully expressed in Escherichia coli for analysis of their biochemical functions. The catalytic activity of our bacterially expressed ancestral ARODs suggests that our ASR was successful. The ancestral AROD that is phylogenetically most remote from a native AROD has the highest thermal stability and substrate promiscuity. Our findings led us to infer that AROD evolved from a highly thermostable and promiscuous LAAO. As an application, we can design artificial ARODs with improved functions compared with those of native ones.

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“…We generated keto acids by l -amino acid oxidase (LAAO) treatment of a commercial growth medium that is primarily comprised of 2 H, 13 C, 15 N-amino acids. LAAO enzymes are found in many organisms (Hossain et al 2014 ), with different specificity for the substrate amino acids (Nuutinen et al 2012 ; Sun et al 2010 ). We chose as the enzyme source a crude snake venom containing LAAO, which can be applied directly to the commercial growth medium (Fig.…”

Section: Introductionmentioning

confidence: 99%

Alpha protons as NMR probes in deuterated proteins

et al. 2019

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We describe a new labeling method that allows for full protonation at the backbone Hα position, maintaining protein side chains with a high level of deuteration. We refer to the method as alpha proton exchange by transamination (α-PET) since it relies on transaminase activity demonstrated here using Escherichia coli expression. We show that α-PET labeling is particularly useful in improving structural characterization of solid proteins by introduction of an additional proton reporter, while eliminating many strong dipolar couplings. The approach benefits from the high sensitivity associated with 1.3 mm samples, more abundant information including Hα resonances, and the narrow proton linewidths encountered for highly deuterated proteins. The labeling strategy solves amide proton exchange problems commonly encountered for membrane proteins when using perdeuteration and backexchange protocols, allowing access to alpha and all amide protons including those in exchange-protected regions. The incorporation of Hα protons provides new insights, as the close Hα–Hα and Hα–HN contacts present in β-sheets become accessible, improving the chance to determine the protein structure as compared with HN–HN contacts alone. Protonation of the Hα position higher than 90% is achieved for Ile, Leu, Phe, Tyr, Met, Val, Ala, Gln, Asn, Thr, Ser, Glu, Asp even though LAAO is only active at this degree for Ile, Leu, Phe, Tyr, Trp, Met. Additionally, the glycine methylene carbon is labeled preferentially with a single deuteron, allowing stereospecific assignment of glycine alpha protons. In solution, we show that the high deuteration level dramatically reduces R2 relaxation rates, which is beneficial for the study of large proteins and protein dynamics. We demonstrate the method using two model systems, as well as a 32 kDa membrane protein, hVDAC1, showing the applicability of the method to study membrane proteins.

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l-Amino acid oxidase of the fungus Hebeloma cylindrosporum displays substrate preference towards glutamate

Cited by 30 publications

References 57 publications

Snake venom L-amino acid oxidases: an overview on their antitumor effects

Snake venom L-amino acid oxidases: an overview on their antitumor effects

Following the Evolutionary Track of a Highly Specific l -Arginine Oxidase by Reconstruction and Biochemical Analysis of Ancestral and Native Enzymes

Alpha protons as NMR probes in deuterated proteins

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