scholarly journals A nematode sterol C4α-methyltransferase catalyzes a new methylation reaction responsible for sterol diversity

2019 ◽  
Vol 61 (2) ◽  
pp. 192-204 ◽  
Author(s):  
Wenxu Zhou ◽  
Paxtyn M. Fisher ◽  
Boden H. Vanderloop ◽  
Yun Shen ◽  
Huazhong Shi ◽  
...  
Keyword(s):  
Metabolic Pathways ◽  
Fossil Record ◽  
Sedimentary Rock ◽  
Amino Acid Sequence Identity ◽  
Dietary Cholesterol ◽  
Specific Substrate ◽  
Metabolism Pathway ◽  
Sequence Identity ◽  
Binding Domains ◽  
Methyl Sterols

Primitive sterol evolution plays an important role in fossil record interpretation and offers potential therapeutic avenues for human disease resulting from nematode infections. Recognizing that C4-methyl stenol products [8(14)-lophenol] can be synthesized in bacteria while C4-methyl stanol products (dinosterol) can be synthesized in dinoflagellates and preserved as sterane biomarkers in ancient sedimentary rock is key to eukaryotic sterol evolution. In this regard, nematodes have been proposed to convert dietary cholesterol to 8(14)-lophenol by a secondary metabolism pathway that could involve sterol C4 methylation analogous to the C2 methylation of hopanoids (radicle-type mechanism) or C24 methylation of sterols (carbocation-type mechanism). Here, we characterized dichotomous cholesterol metabolic pathways in Caenorhabditis elegans that generate 3-oxo sterol intermediates in separate paths to lophanol (4-methyl stanol) and 8(14)-lophenol (4-methyl stenol). We uncovered alternate C3-sterol oxidation and Δ7 desaturation steps that regulate sterol flux from which branching metabolite networks arise, while lophanol/8(14)-lophenol formation is shown to be dependent on a sterol C4α-methyltransferse (4-SMT) that requires 3-oxo sterol substrates and catalyzes a newly discovered 3-keto-enol tautomerism mechanism linked to S-adenosyl-l-methionine-dependent methylation. Alignment-specific substrate-binding domains similarly conserved in 4-SMT and 24-SMT enzymes, despite minimal amino acid sequence identity, suggests divergence from a common, primordial ancestor in the evolution of methyl sterols. The combination of these results provides evolutionary leads to sterol diversity and points to cryptic C4-methyl steroidogenic pathways of targeted convergence that mediate lineage-specific adaptations.­­

Biochemical characterization of a glucoamylase from Saccharomycopsis fibuligera R64

Biologia ◽  
2011 ◽  
Vol 66 (1) ◽  
Author(s):  
Dessy Natalia ◽  
Keni Vidilaseris ◽  
Pasjan Satrimafitrah ◽  
Wangsa Ismaya ◽  
Purkan ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Amino Acid ◽  
Amino Acid Sequence Identity ◽  
Biochemical Characterization ◽  
Saccharomycopsis Fibuligera ◽  
Sequence Identity ◽  
Ic50 Value ◽  
High Amino Acid ◽  
Ph Range

AbstractGlucoamylase from the yeast Saccharomycopsis fibuligera R64 (GLL1) has successfully been purified and characterized. The molecular mass of the enzyme was 56,583 Da as determined by mass spectrometry. The purified enzyme demonstrated optimum activity in the pH range of 5.6–6.4 and at 50°C. The activity of the enzyme was inhibited by acarbose with the IC50 value of 5 μM. GLL1 shares high amino acid sequence identity with GLU1 and GLA1, which are Saccharomycopsis fibuligera glucoamylases from the strains HUT7212 and KZ, respectively. The properties of GLL1, however, resemble that of GLU1. The elucidation of the primary structure of GLL1 contributes to the explanation of this finding.

scholarly journals Petunia vein banding virus: Characterization of a New Tymovirus from Petunia × hybrida

Plant Disease ◽  
2000 ◽  
Vol 84 (7) ◽  
pp. 739-742 ◽  
Author(s):  
M. A. V. Alexandre ◽  
L. M. L. Duarte ◽  
E. B. Rivas ◽  
C. M. Chagas ◽  
M. M. Barradas ◽  
...  
Keyword(s):  
Nicotiana Benthamiana ◽  
Petunia Hybrida ◽  
Amino Acid Sequence Identity ◽  
Rio Grande Do Sul ◽  
Cytopathic Effects ◽  
Sequence Identity ◽  
Andean Potato ◽  
Plant Families ◽  
Nicandra Physalodes

Petunia plants from a nursery in the State of Rio Grande do Sul, Brazil, showed pronounced vein banding and contained isometric particles with diameters of approximately 45 and 30 nm. The larger ones apparently represent a caulimovirus, while the smaller ones, which included both empty shells and full particles, were identified as those of a new tymovirus for which we propose the name Petunia vein banding virus (PetVBV). Originally, PetVBV was transmitted only with difficulty to healthy petunia plants. However, from an experimentally infected petu-nia, it was later readily transmitted also to Nicotiana benthamiana and Nicandra physalodes, but not to other species in the Solanaceae or other plant families. It produces cytopathic effects typical for tymovirus infections. Its coat protein shows approximately 65% amino acid sequence identity with those of Eggplant mosaic and Andean potato latent viruses, to which it is also serologically more closely related than to any other tymoviruses.

scholarly journals Four Novel Mycoviruses from the Hypovirulent Botrytis cinerea SZ-2-3y Isolate from Paris polyphylla: Molecular Characterisation and Mitoviral Sequence Transboundary Entry into Plants

Viruses ◽  
2022 ◽  
Vol 14 (1) ◽  
pp. 151
Author(s):  
Qiong Wang ◽  
Qi Zou ◽  
Zhaoji Dai ◽  
Ni Hong ◽  
Guoping Wang ◽  
...  
Keyword(s):  
Botrytis Cinerea ◽  
Amino Acid Sequence Identity ◽  
Horizontal Transmission ◽  
Plant Mitochondria ◽  
Full Length ◽  
Genome Sequences ◽  
Rna Dependent Rna Polymerase ◽  
New Members ◽  
Sequence Identity ◽  
Paris Polyphylla

A hypovirulent SZ-2-3y strain isolated from diseased Paris polyphylla was identified as Botrytis cinerea. Interestingly, SZ-2-3y was coinfected with a mitovirus, two botouliviruses, and a 3074 nt fusarivirus, designated Botrytis cinerea fusarivirus 8 (BcFV8); it shares an 87.2% sequence identity with the previously identified Botrytis cinerea fusarivirus 6 (BcFV6). The full-length 2945 nt genome sequence of the mitovirus, termed Botrytis cinerea mitovirus 10 (BcMV10), shares a 54% sequence identity with Fusarium boothii mitovirus 1 (FbMV1), and clusters with fungus mitoviruses, plant mitoviruses and plant mitochondria; hence BcMV10 is a new Mitoviridae member. The full-length 2759 nt and 2812 nt genome sequences of the other two botouliviruses, named Botrytis cinerea botoulivirus 18 and 19 (BcBoV18 and 19), share a 40% amino acid sequence identity with RNA-dependent RNA polymerase protein (RdRp), and these are new members of the Botoulivirus genus of Botourmiaviridae. Horizontal transmission analysis showed that BcBoV18, BcBoV19 and BcFV8 are not related to hypovirulence, suggesting that BcMV10 may induce hypovirulence. Intriguingly, a partial BcMV10 sequence was detected in cucumber plants inoculated with SZ-2-3y mycelium or pXT1/BcMV10 agrobacterium. In conclusion, we identified a hypovirulent SZ-2-3y fungal strain from P. polyphylla, coinfected with four novel mycoviruses that could serve as potential biocontrol agents. Our findings provide evidence of cross-kingdom mycoviral sequence transmission.

scholarly journals Nucleotide Sequence and Characterization of a Novel Cefotaxime-Hydrolyzing β-Lactamase (CTX-M-10) Isolated in Spain

2001 ◽  
Vol 45 (2) ◽  
pp. 616-620 ◽  
Author(s):  
Antonio Oliver ◽  
José Claudio Pérez-Dı́az ◽  
Teresa M. Coque ◽  
Fernando Baquero ◽  
Rafael Cantón
Keyword(s):  
Amino Acid ◽  
Nucleotide Sequence ◽  
Amino Acid Sequence Identity ◽  
Penicillin G ◽  
Nucleotide Substitutions ◽  
Sequence Identity ◽  
M 10 ◽  
Hydrolysis Rates ◽  
Kluyvera Ascorbata

ABSTRACT A cefotaxime-resistant, ceftazidime-susceptible Escherichia coli isolate was obtained from a patient with sepsis in 1997, from which a β-lactamase with a pI of 8.1 was cloned. Cephaloridine and cefotaxime relative hydrolysis rates were 167 and 81, respectively (penicillin G rate = 100), whereas ceftazidime hydrolysis was not detected. The nucleotide sequence revealed a bla gene related to that coding for CTX-M-3. Despite 21 nucleotide substitutions, only 2 determined amino acid changes (Ala27Val and Arg38Gln). The amino acid sequence identity between this enzyme, designated CTX-M-10, and the chromosomal β-lactamase ofKluyvera ascorbata was 81%.

scholarly journals Enzyme Specificity of 2-Nitrotoluene 2,3-Dioxygenase from Pseudomonas sp. Strain JS42 Is Determined by the C-Terminal Region of the α Subunit of the Oxygenase Component

1998 ◽  
Vol 180 (5) ◽  
pp. 1194-1199 ◽  
Author(s):  
Juanito V. Parales ◽  
Rebecca E. Parales ◽  
Sol M. Resnick ◽  
David T. Gibson
Keyword(s):  
Amino Acid Sequence Identity ◽  
Large Subunit ◽  
Small Subunit ◽  
Terminal Region ◽  
Oxidation Products ◽  
Enzyme Specificity ◽  
Α Subunit ◽  
Sequence Identity ◽  
Wide Range ◽  
Genes Encoding

ABSTRACT Biotransformations with recombinant Escherichia coliexpressing the genes encoding 2-nitrotoluene 2,3-dioxygenase (2NTDO) from Pseudomonas sp. strain JS42 demonstrated that 2NTDO catalyzes the dihydroxylation and/or monohydroxylation of a wide range of aromatic compounds. Extremely high nucleotide and deduced amino acid sequence identity exists between the components from 2NTDO and the corresponding components from 2,4-dinitrotoluene dioxygenase (2,4-DNTDO) from Burkholderia sp. strain DNT (formerlyPseudomonas sp. strain DNT). However, comparisons of the substrates oxidized by these dioxygenases show that they differ in substrate specificity, regiospecificity, and the enantiomeric composition of their oxidation products. Hybrid dioxygenases were constructed with the genes encoding 2NTDO and 2,4-DNTDO. Biotransformation experiments with these hybrid dioxygenases showed that the C-terminal region of the large subunit of the oxygenase component (ISPα) was responsible for the enzyme specificity differences observed between 2NTDO and 2,4-DNTDO. The small subunit of the terminal oxygenase component (ISPβ) was shown to play no role in determining the specificities of these dioxygenases.

scholarly journals Distinct Regioselectivity of Fungal P450 Enzymes for Steroidal Hydroxylation

2019 ◽  
Vol 85 (18) ◽  
Author(s):  
Wei Lu ◽  
Jinhui Feng ◽  
Xi Chen ◽  
Yun-Juan Bao ◽  
Yu Wang ◽  
...  
Keyword(s):  
Amino Acid ◽  
Pichia Pastoris ◽  
Organic Synthesis ◽  
Transcriptome Sequencing ◽  
Amino Acid Sequence Identity ◽  
Steroid Nucleus ◽  
Content Type ◽  
Whole Cells ◽  
Sequence Identity ◽  
High Amino Acid

ABSTRACT In this study, we identified two P450 enzymes (CYP5150AP3 and CYP5150AN1) from Thanatephorus cucumeris NBRC 6298 by combination of transcriptome sequencing and heterologous expression in Pichia pastoris. The biotransformation of 11-deoxycortisol and testosterone by Pichia pastoris whole cells coexpressing the cyp5150ap3 and por genes demonstrated that the CYP5150AP3 enzyme possessed steroidal 7β-hydroxylase activities toward these substrates, and the regioselectivity was dependent on the structures of steroidal compounds. CYP5150AN1 catalyzed the 2β-hydroxylation of 11-deoxycortisol. It is interesting that they display different regioselectivity of hydroxylation from that of their isoenzyme, CYP5150AP2, which possesses 19- and 11β-hydroxylase activities. IMPORTANCE The steroidal hydroxylases CYP5150AP3 and CYP5150AN1 together with the previously characterized CYP5150AP2 belong to the CYP5150A family of P450 enzymes with high amino acid sequence identity, but they showed completely different regioselectivities toward 11-deoxycortisol, suggesting the regioselectivity diversity of steroidal hydroxylases of CYP5150 family. They are also distinct from the known bacterial and fungal steroidal hydroxylases in substrate specificity and regioselectivity. Biocatalytic hydroxylation is one of the important transformations for the functionalization of steroid nucleus rings but remains a very challenging task in organic synthesis. These hydroxylases are useful additions to the toolbox of hydroxylase enzymes for the functionalization of steroids at various positions.

Immunization of merino ewes with a synthetic inhibin peptide or with preparations obtained from bovine and porcine follicular fluids by immunoaffinity chromatography result in different effects on ovulation rate and on plasma gonadotrophin concentrations

1991 ◽  
Vol 3 (6) ◽  
pp. 659 ◽  
Author(s):  
T O'Shea ◽  
CM Andrews ◽  
BM Bindon ◽  
MA Hillard ◽  
K Miyamoto ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Synthetic Peptide ◽  
Amino Acid Sequence Identity ◽  
Immunoaffinity Chromatography ◽  
Ovulation Rate ◽  
Alpha Subunit ◽  
Ovarian Activity ◽  
Amino Terminal ◽  
Sequence Identity ◽  
Follicular Fluids

Ewes were immunized with either a synthetic peptide (peptide 1-32) that has an amino acid sequence identity with the first 32 amino acids at the amino terminal of the alpha-subunit of porcine inhibin, or with bovine or porcine monoclonal antibody purified inhibin (bMPI and pMPI respectively), obtained by immunochromatography from follicular fluids. The peptide 1-32 was conjugated to albumin before use. Peptide 1-32 and bMPI increased ovulation rate and number of follicles (greater than or equal to 5 mm diameter). Although bMPI increased plasma FSH concentration the peptide did not. pMPI had no effect on ovarian activity but markedly elevated both plasma FSH and LH concentrations. The plasma LH concentration was lowered in ewes immunized with peptide 1-32. It appears, therefore, that ovulation rate can be increased following increased plasma FSH concentrations at luteolysis or in the absence of such an increase. Conversely, greatly increased plasma gonadotrophin concentrations at luteolysis (pMPI) were not followed by an increase in ovulation rate. Antibodies in the plasma of ewes immunized with peptide 1-32 and bMPI bound to iodinated synthetic human inhibin alpha-chain 6-30 peptide. The results suggest that ovulation rate is at least partly determined by intraovarian factors.

Structural features underlying the selective cleavage of a novel exo-type maltose-forming amylase fromPyrococcussp. ST04

2014 ◽  
Vol 70 (6) ◽  
pp. 1659-1668 ◽  
Author(s):  
Kwang-Hyun Park ◽  
Jong-Hyun Jung ◽  
Sung-Goo Park ◽  
Myeong-Eun Lee ◽  
James F. Holden ◽  
...  
Keyword(s):  
Amino Acids ◽  
Molecular Basis ◽  
Amino Acid Sequence Identity ◽  
Structural Features ◽  
Industrial Applications ◽  
Closed Cavity ◽  
Helical Bundles ◽  
Sequence Identity ◽  
Hydrolysis Of ◽  
Potential Use

A novel maltose-forming α-amylase (PSMA) was recently found in the hyperthermophilic archaeonPyrococcussp. ST04. This enzyme shows <13% amino-acid sequence identity to other known α-amylases and displays a unique enzymatic property in that it hydrolyzes both α-1,4-glucosidic and α-1,6-glucosidic linkages of substrates, recognizing only maltose units, in an exo-type manner. Here, the crystal structure of PSMA at a resolution of 1.8 Å is reported, showing a tight ring-shaped tetramer with monomers composed of two domains: an N-domain (amino acids 1–341) with a typical GH57 family (β/α)7-barrel fold and a C-domain (amino acids 342–597) composed of α-helical bundles. A small closed cavity observed in proximity to the catalytic residues Glu153 and Asp253 at the domain interface has the appropriate volume and geometry to bind a maltose unit, accounting for the selective exo-type maltose hydrolysis of the enzyme. A narrow gate at the putative subsite +1 formed by residue Phe218 and Phe452 is essential for specific cleavage of glucosidic bonds. The closed cavity at the active site is connected to a short substrate-binding channel that extends to the central hole of the tetramer, exhibiting a geometry that is significantly different from classical maltogenic amylases or β-amylases. The structural features of this novel exo-type maltose-forming α-amylase provide a molecular basis for its unique enzymatic characteristics and for its potential use in industrial applications and protein engineering.

scholarly journals S-Inosyl-l-Homocysteine Hydrolase, a Novel Enzyme Involved inS-Adenosyl-l-Methionine Recycling

2015 ◽  
Vol 197 (14) ◽  
pp. 2284-2291 ◽  
Author(s):  
Danielle Miller ◽  
Huimin Xu ◽  
Robert H. White
Keyword(s):  
Metabolic Pathways ◽  
Methanogenic Archaea ◽  
Metabolite Identification ◽  
Strictly Anaerobic ◽  
Methanocaldococcus Jannaschii ◽  
Content Type ◽  
Sequence Identity ◽  
Feedback Inhibitor ◽  
Recycling Pathway ◽  
Insight Into

ABSTRACTS-Adenosyl-l-homocysteine, the product ofS-adenosyl-l-methionine (SAM) methyltransferases, is known to be a strong feedback inhibitor of these enzymes. A hydrolase specific forS-adenosyl-l-homocysteine producesl-homocysteine, which is remethylated to methionine and can be used to regenerate SAM. Here, we show that the annotatedS-adenosyl-l-homocysteine hydrolase inMethanocaldococcus jannaschiiis specific for the hydrolysis and synthesis ofS-inosyl-l-homocysteine, notS-adenosyl-l-homocysteine. This is the first report of an enzyme specific forS-inosyl-l-homocysteine. As withS-adenosyl-l-homocysteine hydrolase, which shares greater than 45% sequence identity with theM. jannaschiihomologue, theM. jannaschiienzyme was found to copurify with bound NAD+and hasKmvalues of 0.64 ± 0.4 mM, 0.0054 ± 0.006 mM, and 0.22 ± 0.11 mM for inosine,l-homocysteine, andS-inosyl-l-homocysteine, respectively. No enzymatic activity was detected withS-adenosyl-l-homocysteine as the substrate in either the synthesis or hydrolysis direction. These results prompted us to redesignate theM. jannaschiienzyme anS-inosyl-l-homocysteine hydrolase (SIHH). Identification of SIHH demonstrates a modified pathway in this methanogen for the regeneration of SAM fromS-adenosyl-l-homocysteine that uses the deamination ofS-adenosyl-l-homocysteine to formS-inosyl-l-homocysteine.IMPORTANCEIn strictly anaerobic methanogenic archaea, such asMethanocaldococcus jannaschii, canonical metabolic pathways are often not present, and instead, unique pathways that are deeply rooted on the phylogenetic tree are utilized by the organisms. Here, we discuss the recycling pathway forS-adenosyl-l-homocysteine, produced fromS-adenosyl-l-methionine (SAM)-dependent methylation reactions, which uses a hydrolase specific forS-inosyl-l-homocysteine, an uncommon metabolite. Identification of the pathways and the enzymes involved in the unique pathways in the methanogens will provide insight into the biochemical reactions that were occurring when life originated.

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