Pyridoxal-5′-phosphate as an oxygenase cofactor: Discovery of a carboxamide-forming, α-amino acid monooxygenase-decarboxylase

Capuramycins are antimycobacterial antibiotics that consist of a modified nucleoside named uridine-5′-carboxamide (CarU). Previous biochemical studies have revealed that CarU is derived from UMP, which is first converted to uridine-5′-aldehyde in a reaction catalyzed by the dioxygenase CapA and subsequently to 5′-C-glycyluridine (GlyU), an unusual β–hydroxy-α-amino acid, in a reaction catalyzed by the pyridoxal-5′-phosphate (PLP)-dependent transaldolase CapH. The remaining steps that are necessary to furnish CarU include decarboxylation, O atom insertion, and oxidation. We demonstrate that Cap15, which has sequence similarity to proteins annotated as bacterial, PLP-dependent l-seryl-tRNA(Sec) selenium transferases, is the sole catalyst responsible for complete conversion of GlyU to CarU. Using a complementary panel of in vitro assays, Cap15 is shown to be dependent upon substrates O2 and (5′S,6′R)-GlyU, the latter of which was unexpected given that (5′S,6′S)-GlyU is the isomeric product of the transaldolase CapH. The two products of Cap15 are identified as the carboxamide-containing CarU and CO2. While known enzymes that catalyze this type of chemistry, namely α-amino acid 2-monooxygenase, utilize flavin adenine dinucleotide as the redox cofactor, Cap15 remarkably requires only PLP. Furthermore, Cap15 does not produce hydrogen peroxide and is shown to directly incorporate a single O atom from O2 into the product CarU and thus is an authentic PLP-dependent monooxygenase. In addition to these unusual discoveries, Cap15 activity is revealed to be dependent upon the inclusion of phosphate. The biochemical characteristics along with initiatory mechanistic studies of Cap15 are reported, which has allowed us to assign Cap15 as a PLP-dependent (5′S,6′R)-GlyU:O2 monooxygenase-decarboxylase.

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Genetic Characterization of Avian Influenza A (H11N9) Virus Isolated from Mandarin Ducks in South Korea in 2018

Viruses ◽

10.3390/v12020203 ◽

2020 ◽

Vol 12 (2) ◽

pp. 203

Author(s):

Hien Thi Tuong ◽

Ngoc Minh Nguyen ◽

Haan Woo Sung ◽

Hyun Park ◽

Seon-Ju Yeo

Keyword(s):

Influenza Virus ◽

Amino Acid ◽

Avian Influenza ◽

South Korea ◽

Avian Influenza Virus ◽

Influenza A ◽

Sequence Similarity ◽

Basic Amino Acid ◽

Wild Birds

In July 2018, a novel avian influenza virus (A/Mandarin duck/South Korea/KNU18-12/2018(H11N9)) was isolated from Mandarin ducks in South Korea. Phylogenetic and molecular analyses were conducted to characterize the genetic origins of the H11N9 strain. Phylogenetic analysis indicated that eight gene segments of strain H11N9 belonged to the Eurasian lineages. Analysis of nucleotide sequence similarity of both the hemagglutinin (HA) and neuraminidase (NA) genes revealed the highest homology with A/duck/Kagoshima/KU57/2014 (H11N9), showing 97.70% and 98.00% nucleotide identities, respectively. Additionally, internal genes showed homology higher than 98% compared to those of other isolates derived from duck and wild birds. Both the polymerase acidic (PA) and polymerase basic 1 (PB1) genes were close to the H5N3 strain isolated in China; whereas, other internal genes were closely related to that of avian influenza virus in Japan. A single basic amino acid at the HA cleavage site (PAIASR↓GLF), the lack of a five-amino acid deletion (residue 69–73) in the stalk region of the NA gene, and E627 in the polymerase basic 2 (PB2) gene indicated that the A/Mandarin duck/South Korea/KNU18-12/2018(H11N9) isolate was a typical low-pathogenicity avian influenza. In vitro viral replication of H11N9 showed a lower titer than H1N1 and higher than H9N2. In mice, H11N9 showed lower adaptation than H1N1. The novel A/Mandarin duck/South Korea/KNU18-12/2018(H11N9) isolate may have resulted from an unknown reassortment through the import of multiple wild birds in Japan and Korea in approximately 2016–2017, evolving to produce a different H11N9 compared to the previous H11N9 in Korea (2016). Further reassortment events of this virus occurred in PB1 and PA in China-derived strains. These results indicate that Japanese- and Chinese-derived avian influenza contributes to the genetic diversity of A/Mandarin duck/South Korea/KNU18-12/2018(H11N9) in Korea.

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The Pseudoalteromonas luteoviolacea L-amino Acid Oxidase with Antimicrobial Activity Is a Flavoenzyme

Marine Drugs ◽

10.3390/md16120499 ◽

2018 ◽

Vol 16 (12) ◽

pp. 499 ◽

Cited By ~ 3

Author(s):

Andrés Andreo-Vidal ◽

Antonio Sanchez-Amat ◽

Jonatan Campillo-Brocal

Keyword(s):

Antimicrobial Activity ◽

Amino Acid ◽

Flavin Adenine Dinucleotide ◽

Sequence Similarity ◽

Bacterial Species ◽

Biological Significance ◽

Acid Oxidase ◽

Biotechnological Applications ◽

Amino Acid Oxidase ◽

First Time

The marine environment is a rich source of antimicrobial compounds with promising pharmaceutical and biotechnological applications. The Pseudoalteromonas genus harbors one of the highest proportions of bacterial species producing antimicrobial molecules. For decades, the presence of proteins with L-amino acid oxidase (LAAO) and antimicrobial activity in Pseudoalteromonas luteoviolacea has been known. Here, we present for the first time the identification, cloning, characterization and phylogenetic analysis of Pl-LAAO, the enzyme responsible for both LAAO and antimicrobial activity in P. luteoviolacea strain CPMOR-2. Pl-LAAO is a flavoprotein of a broad substrate range, in which the hydrogen peroxide generated in the LAAO reaction is responsible for the antimicrobial activity. So far, no protein with a sequence similarity to Pl-LAAO has been cloned or characterized, with this being the first report on a flavin adenine dinucleotide (FAD)-containing LAAO with antimicrobial activity from a marine microorganism. Our results revealed that 20.4% of the sequenced Pseudoalteromonas strains (specifically, 66.6% of P. luteoviolacea strains) contain Pl-laao similar genes, which constitutes a well-defined phylogenetic group. In summary, this work provides insights into the biological significance of antimicrobial LAAOs in the Pseudoalteromonas genus and shows an effective approach for the detection of novel LAAOs, whose study may be useful for biotechnological applications.

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Antagonism of Trichoderma harzianum ETS 323 on Botrytis cinerea Mycelium in Culture Conditions

Phytopathology ◽

10.1094/phyto-11-11-0315 ◽

2012 ◽

Vol 102 (11) ◽

pp. 1054-1063 ◽

Cited By ~ 23

Author(s):

Chi-Hua Cheng ◽

Chia-Ann Yang ◽

Kou-Cheng Peng

Keyword(s):

Amino Acid ◽

Botrytis Cinerea ◽

Trichoderma Harzianum ◽

Hyphal Growth ◽

Extracellular Proteins ◽

In Vitro Assays ◽

Acid Oxidase ◽

Amino Acid Oxidase ◽

Trichoderma Spp

Previous studies have shown that the extracellular proteins of Trichoderma harzianum ETS 323 grown in the presence of deactivated Botrytis cinerea in culture include a putative l-amino acid oxidase and have suggested the involvement of this enzyme in the antagonistic mechanism. Here, we hypothesized that the mycoparasitic process of Trichoderma spp. against B. cinerea involves two steps; that is, an initial hyphal coiling stage and a subsequent hyphal coiling stage, with different coiling rates. The two-step antagonism of T. harzianum ETS 323 against B. cinerea during the mycoparasitic process in culture was evaluated using a biexponential equation. In addition, an l-amino acid oxidase (Th-l-AAO) was identified from T. harzianum ETS 323. The secretion of Th-l-AAO was increased when T. harzianum ETS 323 was grown with deactivated hyphae of B. cinerea. Moreover, in vitro assays indicated that Th-l-AAO effectively inhibited B. cinerea hyphal growth, caused cytosolic vacuolization in the hyphae, and led to hyphal lysis. Th-l-AAO also showed disease control against the development of B. cinerea on postharvest apple fruit and tobacco leaves. Furthermore, an apoptosis-like response, including the generation of reactive oxygen species, was observed in B. cinerea after treatment with Th-l-AAO, suggesting that Th-l-AAO triggers programmed cell death in B. cinerea. This may be associated with the two-step antagonism of T. harzianum ETS 323 against B. cinerea.

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Human carboxypeptidase E. Isolation and characterization of the cDNA, sequence conservation, expression and processing in vitro

Biochemical Journal ◽

10.1042/bj2670517 ◽

1990 ◽

Vol 267 (2) ◽

pp. 517-525 ◽

Cited By ~ 44

Author(s):

E Manser ◽

D Fernandez ◽

L Loo ◽

P Y Goh ◽

C Monfries ◽

...

Keyword(s):

Amino Acid ◽

Membrane Protein ◽

Sequence Similarity ◽

Coding Region ◽

Sequence Comparisons ◽

Brain Membrane ◽

Carboxypeptidase E ◽

Isolation And Characterization ◽

Microsomal Membranes

Carboxypeptidase E (CPE), which cleaves C-terminal amino acid residues and is involved in neuropeptide processing, is itself subject to intracellular processing. Human CPE cDNA was isolated and sequence comparisons were made with those of a previously isolated brain cDNA (M1622) encoding rat CPE and of other human carboxypeptidases (M and N). Human (2.5 kb) and rat (2.1 kb) CPE cDNAs approximated to the size of their respective mRNAs; additional sequences were located in putative 5′ and 3′ untranslated regions of human CPE mRNA. There is 79% sequence similarity between human and rat CPE cDNAs, with greater similarity (89%) over the coding region and short sections of the non-coding sequence. The predicted 476-amino acid-residue sequences of human and rat preproCPEs are highly conserved (96% identity), with lower degree of similarity of the N-terminal signal peptide (76%). Human CPE showed 51% and 43% sequence similarity to human CPN and CPM respectively, with discrete regions of divergence dispersed between the highly conserved mechanistically implicated regions. Antiserum generated from a fusion protein, synthesized in Escherichia coli from constructs of the human cDNA, recognized an approx. 50 kDa membrane protein and a smaller soluble protein in rat and human brain preparations, corresponding to the two forms of native CPE. Human CPE mRNA transcripts directed the synthesis in reticulocyte lysate of a 54 kDa translation product, which in the presence of dog pancreas microsomal membranes was co-translationally processed with cleavage, insertion into membranes and glycosylation. Three processed forms were generated, the largest (56 kDa) and smallest (52 kDa) being equally glycosylated. The membrane association of the processed translation products and of native brain membrane CPE, detected immunologically, was resistant to moderate alkali but not pH 11.5 extraction. These results are consistent with secondary-structure predictions that CPE is a peripheral membrane protein. The dissimilar regions of human carboxypeptidases may provide information on sequences responsible for their different cellular disposition.

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THE POLYCYSTIC OVARY. VI.

Acta Endocrinologica ◽

10.1530/acta.0.0520425 ◽

1966 ◽

Vol 52 (3) ◽

pp. 425-442 ◽

Cited By ~ 13

Author(s):

Jesus Corral-Gallardo ◽

Hernan A. Acevedo ◽

Jose L. Perez de Salazar ◽

Manuel Loria ◽

Joseph W. Goldzieher

Keyword(s):

Polycystic Ovary ◽

Ovarian Tissue ◽

Hydroxysteroid Dehydrogenase ◽

Cell Tumour ◽

Primary Amenorrhea ◽

Biochemical Characteristics ◽

Testicular Tumours ◽

Biochemical Studies

ABSTRACT Detailed clinical and biochemical studies were performed in a 22 year old woman with primary amenorrhea, virilization, bilateral sclerocystic ovarian disease and a hilar cell tumour of one ovary. By clinical, radiological and dynamic functional endocrine tests, the ovarian source of the excessive androgen production was established prior to surgery. In vivo and in vitro production of testosterone by the tumour was established as well as its ability to metabolize progesterone and 4-androstenedione to a variety of compounds. The biochemical characteristics of this tumour strongly resemble testicular tumours, even to the existence of an 11β-hydroxylase. Epitestosterone was found in the urine, and the existence of a 17α-hydroxysteroid dehydrogenase was demonstrated in the sclerocystic ovarian tissue. No oestrogen was synthesized by the tumour or the ovarian tissue under the in vitro conditions.

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The penicillin binding protein 1A of Helicobacter pylori, its amoxicillin binding site and access routes

Gut Pathogens ◽

10.1186/s13099-021-00438-0 ◽

2021 ◽

Vol 13 (1) ◽

Author(s):

Bahareh Attaran ◽

Najmeh Salehi ◽

Bahareh Ghadiri ◽

Maryam Esmaeili ◽

Shadi Kalateh ◽

...

Keyword(s):

Amino Acid ◽

Binding Site ◽

3D Structure ◽

Resistance Mechanisms ◽

Drug Binding ◽

In Vitro Assays ◽

Amino Acid Residues ◽

Clinical Strains ◽

H Pylori

Abstract Background Amoxicillin-resistant H. pylori strains are increasing worldwide. To explore the potential resistance mechanisms involved, the 3D structure modeling and access tunnel prediction for penicillin-binding proteins (PBP1A) was performed, based on the Streptococcus pneumoniae, PBP 3D structure. Molecular covalent docking was used to determine the interactions between amoxicillin (AMX) and PBP1A. Results The AMX-Ser368 covalent complex interacts with the binding site residues (Gly367, Ala369, ILE370, Lys371, Tyr416, Ser433, Thr541, Thr556, Gly557, Thr558, and Asn560) of PBP1A, non-covalently. Six tunnel-like structures, accessing the PBP1A binding site, were characterized, using the CAVER algorithm. Tunnel-1 was the ultimate access route, leading to the drug catalytic binding residue (Ser368). This tunnel comprises of eighteen amino acid residues, 8 of which are shared with the drug binding site. Subsequently, to screen the presence of PBP1A mutations, in the binding site and tunnel residues, in our clinical strains, in vitro assays were performed. H. pylori strains, isolated under gastroscopy, underwent AMX susceptibility testing by E-test. Of the 100 clinical strains tested, 4 were AMX-resistant. The transpeptidase domain of the pbp1a gene of these resistant, plus 10 randomly selected AMX-susceptible strains, were amplified and sequenced. Of the amino acids lining the tunnel-1 and binding site residues, three (Ser414Arg, Val469Met and Thr556Ser) substitutions, were detected in 2 of the 4 resistant and none of the sequenced susceptible strains, respectively. Conclusions We hypothesize that mutations in amino acid residues lining the binding site and/or tunnel-1, resulting in conformational/spatial changes, may block drug binding to PBP1A and cause AMX resistance.

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Feedback Regulation of N Fixation in Frankia-Alnus Symbiosis Through Amino Acids Profiling in Field and Greenhouse Nodules

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-10-19-0289-r ◽

2020 ◽

Vol 33 (3) ◽

pp. 499-508

Author(s):

Anne-Emmanuelle Hay ◽

Aude Herrera-Belaroussi ◽

Marjolaine Rey ◽

Pascale Fournier ◽

Philippe Normand ◽

...

Keyword(s):

Amino Acids ◽

Nitrogen Fixation ◽

Amino Acid ◽

Feedback Regulation ◽

Metabolic Model ◽

In Vitro Assays ◽

N Fixation ◽

Abundance Pattern ◽

High Level

Symbiosis established between actinorhizal plants and Frankia spp., which are nitrogen-fixing actinobacteria, promotes nodule organogenesis, the site of metabolic exchange. The present study aimed to identify amino acid markers involved in Frankia-Alnus interactions by comparing nodules and associated roots from field and greenhouse samples. Our results revealed a high level of citrulline in all samples, followed by arginine (Arg), aspartate (Asp), glutamate (Glu), γ-amino-n-butyric acid (GABA), and alanine (Ala). Interestingly, the field metabolome approach highlighted more contrasted amino acid patterns between nodules and roots compared with greenhouse samples. Indeed, 12 amino acids had a mean relative abundance significantly different between field nodule and root samples, against only four amino acids in greenhouse samples, underlining the importance of developing “ecometabolome” approaches. In order to monitor the effects on Frankia cells (respiration and nitrogen fixation activities) of amino acid with an abundance pattern evocative of a role in symbiosis, in-vitro assays were performed by supplementing them in nitrogen-free cultures. Amino acids had three types of effects: i) those used by Frankia as nitrogen source (Glu, Gln, Asp), ii) amino acids stimulating both nitrogen fixation and respiration (e.g., Cit, GABA, Ala, valine, Asn), and iii) amino acids triggering a toxic effect (Arg, histidine). In this paper, a N-metabolic model was proposed to discuss how the host plant and bacteria modulate amino acids contents in nodules, leading to a fine regulation sustaining high bacterial nitrogen fixation.

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Sinorhizobium meliloti Functionally Replaces 3-Oxoacyl-Acyl Carrier Protein Reductase (FabG) by Overexpressing NodG During Fatty Acid Synthesis

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-07-15-0148-r ◽

2016 ◽

Vol 29 (6) ◽

pp. 458-467 ◽

Cited By ~ 5

Author(s):

Ya-Hui Mao ◽

Feng Li ◽

Jin-Cheng Ma ◽

Zhe Hu ◽

Hai-Hong Wang

Keyword(s):

Fatty Acid ◽

Fatty Acid Synthesis ◽

Sinorhizobium Meliloti ◽

Acyl Carrier Protein ◽

Sequence Similarity ◽

Acid Synthesis ◽

In Vitro Assays ◽

Temperature Sensitive ◽

E Coli

In Sinorhizobium meliloti, the nodG gene is located in the nodFEG operon of the symbiotic plasmid. Although strong sequence similarity (53% amino acid identities) between S. meliloti NodG and Escherichia coli FabG was reported in 1992, it has not been determined whether S. meliloti NodG plays a role in fatty acid synthesis. We report that expression of S. meliloti NodG restores the growth of the E. coli fabG temperature-sensitive mutant CL104 under nonpermissive conditions. Using in vitro assays, we demonstrated that NodG is able to catalyze the reduction of the 3-oxoacyl-ACP intermediates in E. coli fatty acid synthetic reaction. Moreover, although deletion of the S. meliloti nodG gene does not cause any growth defects, upon overexpression of nodG from a plasmid, the S. meliloti fabG gene encoding the canonical 3-oxoacyl-ACP reductase (OAR) can be disrupted without any effects on growth or fatty acid composition. This indicates that S. meliloti nodG encodes an OAR and can play a role in fatty acid synthesis when expressed at sufficiently high levels. Thus, a bacterium can simultaneously possess two or more OARs that can play a role in fatty acid synthesis. Our data also showed that, although SmnodG increases alfalfa nodulation efficiency, it is not essential for alfalfa nodulation.

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Conformational and Biological Properties of Partial Sequences of Glucagon

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y73-037 ◽

1973 ◽

Vol 51 (4) ◽

pp. 243-248 ◽

Cited By ~ 17

Author(s):

Richard M. Epand ◽

Vijaylaxmi Grey

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Biological Properties ◽

In Vitro Assays ◽

Adenyl Cyclase Activity ◽

Amino Acid Fragment ◽

Acid Fragment ◽

Carboxyl Terminal ◽

Derivatives Of

We have investigated the properties of derivatives of the 29-amino-acid polypeptide hormone, glucagon, which had varying numbers of amino acids cleaved from the carboxyl terminal portion of the molecule. These derivatives included a 27-amino-acid fragment made by cleavage of the native molecule with cyanogen bromide and a 23-amino-acid fragment made synthetically. We found both of these derivatives capable of stimulating the conversion of ATP to cyclic AMP in in vitro assays using rat liver homogenates. The concentration of these peptides, which were required to produce enhanced adenyl cyclase activity, was higher than that of glucagon, and in the case of the 1–23 derivative it was several orders of magnitude larger. The requirement for higher concentrations of peptide is expected, as removal of a large portion of the total number of amino acids will proportionately decrease the free energy of dissociation of the peptide from the membrane receptor, thus changing the equilibrium constant for binding by several orders of magnitude. Circular dichroism and ultra-centrifuge studies of these peptides, as well as a 21-amino-acid fragment made by cleavage of the native molecule with carboxypeptidase, indicated that the shorter glucagon analogues have structures similar to that of the native molecule although somewhat less ordered.

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Aplysia allatotropin-related peptide and its newly identified d-amino acid–containing epimer both activate a receptor and a neuronal target

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.004367 ◽

2018 ◽

Vol 293 (43) ◽

pp. 16862-16873 ◽

Cited By ~ 8

Author(s):

James W. Checco ◽

Guo Zhang ◽

Wang-ding Yuan ◽

Zi-wei Le ◽

Jian Jing ◽

...

Keyword(s):

Amino Acid ◽

Extracellular Space ◽

Sequence Similarity ◽

Model Organism ◽

Aminopeptidase Activity ◽

Primary Role ◽

Cone Snail ◽

Post Translational Modification ◽

Related Peptide

l- to d-residue isomerization is a post-translational modification (PTM) present in neuropeptides, peptide hormones, and peptide toxins from several animals. In most cases, the d-residue is critical for the biological function of the resulting d-amino acid–containing peptide (DAACP). Here, we provide an example in native neuropeptides in which the DAACP and its all-l-amino acid epimer are both active at their newly identified receptor in vitro and at a neuronal target associated with feeding behavior. On the basis of sequence similarity to a known DAACP from cone snail venom, we hypothesized that allatotropin-related peptide (ATRP), a neuropeptide from the neuroscience model organism Aplysia californica, may form multiple diastereomers in the Aplysia central nervous system. We determined that ATRP exists as a d-amino acid–containing peptide (d2-ATRP) and identified a specific G protein–coupled receptor as an ATRP receptor. Interestingly, unlike many previously reported DAACPs and their all-l-residue analogs, both l-ATRP and d2-ATRP were potent agonists of this receptor and active in electrophysiological experiments. Finally, d2-ATRP was much more stable than its all-l-residue counterpart in Aplysia plasma, suggesting that in the case of ATRP, the primary role of the l- to d-residue isomerization may be to protect this peptide from aminopeptidase activity in the extracellular space. Our results indicate that l- to d-residue isomerization can occur even in an all-l-residue peptide with a known biological activity and that in some cases, this PTM may help modulate peptide signal lifetime in the extracellular space rather than activity at the cognate receptor.

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