scholarly journals L-DOPA dioxygenase of the fly agaric toadstool: revision of the dodA gene sequence and mechanism of enzymatic pigment production

2020 ◽  
Author(s):  
Douglas M. M. Soares ◽  
Letícia C. P. Gonçalves ◽  
Caroline O. Machado ◽  
Larissa Cerrato Esteves ◽  
Cassius V. Stevani ◽  
...  
Keyword(s):  
Environmental Remediation ◽  
Kinetic Modelling ◽  
Enzymatic Catalysis ◽  
Heme Iron ◽  
Start Codon ◽  
Amino Acid Residues ◽  
Coding Region ◽  
Biotechnological Production ◽  
Pigment Formation ◽  
Alternative Start Codon

ABSTRACTl-DOPA extradiol dioxygenases (DODAs) catalyze the production of betalains and hygroaurins pigments. The sequence of the DODAs found in Caryophyllales and Basidiomycetes are not conserved, although betalains are produced both by plants and fungi. Here we revise the coding region of the dodA gene of fly agaric [Amanita muscaria (L.) Lam.] and describe an alternative start codon downstream that enables the heterologous expression of AmDODA, a promiscuous l-DOPA dioxygenase. AmDODA is 43-amino acid residues shorter than the recombinant DODA previously reported but catalyzes the formation of two isomeric seco-DOPAs that are the biosynthetic precursors of betalains and hygroaurins. The putative active site of AmDODA contains two distinct His-His-Glu motifs that can explain the dual cleavage of l-DOPA according to the mechanism proposed for non-heme iron-dependent dioxygenases. Upon addition of excess l-DOPA, both the betaxanthin and hygroaurin adducts of l-DOPA are produced. The kinetic parameters of enzymatic catalysis at pH 8.5 are similar to those reported for other l-DOPA dioxygenases. The rate constants for the conversion of l-DOPA into the betalamic acid and muscaflavin were estimated by kinetic modelling allowing the proposal of a mechanism of pigment formation. These results contribute to understanding the biosynthesis of bacterial, fungal and plant pigments, for the biotechnological production of hygroaurins, and for the development of more promiscuous dioxygenases for environmental remediation.

Cloning and analysis of a gene involved in DNA repair and recombination, the rad1 gene of Schizosaccharomyces pombe

1990 ◽  
Vol 10 (7) ◽  
pp. 3750-3760
Author(s):  
P Sunnerhagen ◽  
B L Seaton ◽  
A Nasim ◽  
S Subramani
Keyword(s):  
Dna Repair ◽  
Schizosaccharomyces Pombe ◽  
Protein Product ◽  
Start Codon ◽  
Insertion Mutagenesis ◽  
Amino Acid Residues ◽  
Flanking Sequence ◽  
Coding Region ◽  
Base Pairs ◽  
Repair Gene

We have cloned the rad1 gene of Schizosaccharomyces pombe by complementation of the rad1-1 mutant, which is deficient in DNA repair and recombination. The coding region of the gene is 582 base pairs long and contains no introns. The predicted product is a strongly acidic, 22-kilodalton protein containing 194 amino acid residues. This gene does not exhibit significant homology to any other known repair gene. The major transcription start site is at 27 base pairs upstream of the putative start codon. Insertion mutagenesis revealed that besides the coding region, at least 151 base pairs of 5'-flanking sequence are required for full complementing activity. A strain carrying a null allele of rad1 was constructed and found to have a phenotype closely similar to that of the rad1-1 mutant. Expression in Escherichia coli of the coding region yielded a protein product of a size close to that predicted from the DNA sequence. This product reacted with antibodies raised against a synthetic peptide with a sequence from that predicted for the protein product. We have localized the rad1 gene to NotI fragment E of the S. pombe genome.

scholarly journals Cloning and analysis of a gene involved in DNA repair and recombination, the rad1 gene of Schizosaccharomyces pombe.

1990 ◽  
Vol 10 (7) ◽  
pp. 3750-3760 ◽  
Author(s):  
P Sunnerhagen ◽  
B L Seaton ◽  
A Nasim ◽  
S Subramani
Keyword(s):  
Dna Repair ◽  
Schizosaccharomyces Pombe ◽  
Protein Product ◽  
Start Codon ◽  
Insertion Mutagenesis ◽  
Amino Acid Residues ◽  
Flanking Sequence ◽  
Coding Region ◽  
Base Pairs ◽  
Repair Gene

We have cloned the rad1 gene of Schizosaccharomyces pombe by complementation of the rad1-1 mutant, which is deficient in DNA repair and recombination. The coding region of the gene is 582 base pairs long and contains no introns. The predicted product is a strongly acidic, 22-kilodalton protein containing 194 amino acid residues. This gene does not exhibit significant homology to any other known repair gene. The major transcription start site is at 27 base pairs upstream of the putative start codon. Insertion mutagenesis revealed that besides the coding region, at least 151 base pairs of 5'-flanking sequence are required for full complementing activity. A strain carrying a null allele of rad1 was constructed and found to have a phenotype closely similar to that of the rad1-1 mutant. Expression in Escherichia coli of the coding region yielded a protein product of a size close to that predicted from the DNA sequence. This product reacted with antibodies raised against a synthetic peptide with a sequence from that predicted for the protein product. We have localized the rad1 gene to NotI fragment E of the S. pombe genome.

scholarly journals Targeted misexpression of NAC052, acting in H3K4 demethylation, alters leaf morphological and anatomical traits in Arabidopsis thaliana

2019 ◽  
Vol 71 (4) ◽  
pp. 1434-1448
Author(s):  
Roxanne van Rooijen ◽  
Stefanie Schulze ◽  
Patrick Petzsch ◽  
Peter Westhoff
Keyword(s):  
Arabidopsis Thaliana ◽  
Leaf Development ◽  
Bundle Sheath ◽  
Start Codon ◽  
Coding Region ◽  
C4 Species ◽  
Alternative Start Codon ◽  
Chlorotic Leaf ◽  
Flaveria Trinervia ◽  
Genetic Regulator

Abstract In an effort to identify genetic regulators for the cell ontogeny around the veins in Arabidopsis thaliana leaves, an activation-tagged mutant line with altered leaf morphology and altered bundle sheath anatomy was characterized. This mutant had a small rosette area with wrinkled leaves and chlorotic leaf edges, as well as enhanced chloroplast numbers in the (pre-)bundle sheath tissue. It had a bundle-specific promoter from the gene GLYCINE DECARBOXYLASE SUBUNIT-T from the C4 species Flaveria trinervia (GLDTFt promoter) inserted in the coding region of the transcriptional repressor NAC052, functioning in H3K4 demethylation, in front of an alternative start codon in-frame with the natural start codon. Reconstruction of the mutation event of our activation-tagged line by creating a line expressing an N-terminally truncated sequence of NAC052 under control of the GLDTFt promoter confirmed the involvement of NAC052 in leaf development. Our study not only reveals leaf anatomic and transcriptomic effects of an N-terminally truncated NAC052 under control of the GLDTFt promoter, but also identifies NAC052 as a novel genetic regulator of leaf development.

scholarly journals Bile-pigment formation from different leghaemoglobins. Methine-bridge specificity of coupled oxidation

1978 ◽  
Vol 176 (2) ◽  
pp. 359-364 ◽  
Author(s):  
Päivi Lehtovaara ◽  
Ulla Perttilä
Keyword(s):  
Amino Acid ◽  
Polypeptide Chain ◽  
Broad Bean ◽  
Amino Acid Sequences ◽  
Second Step ◽  
Bile Pigment ◽  
Site Specificity ◽  
Amino Acid Residues ◽  
Reaction Step ◽  
Pigment Formation

The coupled oxidation of leghaemoglobins with O2 and ascorbate yielded oxyleghaemoglobin in the first reaction step, and the second step was the degradation of haem characterized by an A675 increase. Leghaemoglobins were degraded to biliverdin isomers specifically, depending on the structure of the protein. The main leghaemoglobin components of Glycine (soya bean) and Phaseolus (kidney bean) were degraded to biliverdin mixtures containing about 50% of the β-form, about 30% of the α-form and about 20% of the δ-isomer, whereas the leghaemoglobin I components of Vicia (broad bean) and Pisum (pea) were degraded almost exclusively to the β-isomer, with traces of the α-isomer. The amino acid sequences of Glycine and Phaseolus leghaemoglobins resemble each other, as do those of Vicia and Pisum. The site specificity of bile-pigment formation from leghaemoglobins can be tentatively explained by specific differences in the amino acid sequences at those regions of the polypeptide chain that are in the vicinity of the appropriate methine bridges. The ligand-binding site in different leghaemoglobins may be outlined on the basis of the present results, supposing that the haem is degraded when a reduction product of haem-bound O2 reacts with a methine bridge of the haem, and that the bridge specificity is regulated by hindering amino acid residues that determine the location of the bound O2. The residue phenylalanine-CD1 appears to be further away from the haem plane or in a markedly more flexible position in leghaemoglobins than in mammalian globins. The haem-bound oxygen atom B, in Fe–O(A)–O(B), seems to be free to rotate in all directions except that of the γ-bridge in Glycine and Phaseolus leghaemoglobins, but its position in Vicia and Pisum leghaemoglobin I might be restricted to the direction of the β-methine bridge.

scholarly journals Distinct Subregions of Swi1 Manifest Striking Differences in Prion Transmission and SWI/SNF Function

2010 ◽  
Vol 30 (19) ◽  
pp. 4644-4655 ◽  
Author(s):  
Zhiqiang Du ◽  
Emily T. Crow ◽  
Hyun Seok Kang ◽  
Liming Li
Keyword(s):  
Chromatin Remodeling ◽  
De Novo ◽  
Amino Acid Residues ◽  
Coding Region ◽  
Conformational Switch ◽  
Amyloid Fibers ◽  
Aggregation Pattern ◽  
Prion Transmission ◽  
Shed Light

ABSTRACT We have recently reported that the yeast chromatin-remodeling factor Swi1 can exist as a prion, [SWI +], demonstrating a link between prionogenesis and global transcriptional regulation. To shed light on how the Swi1 conformational switch influences Swi1 function and to define the sequence and structural requirements for [SWI +] formation and propagation, we functionally dissected the Swi1 molecule. We show here that the [SWI +] prion features are solely attributable to the first 327 amino acid residues (N), a region that is asparagine rich. N was aggregated in [SWI+ ] cells but diffuse in [swi− ] cells; chromosomal deletion of the N-coding region resulted in [SWI +] loss, and recombinant N peptide was able to form infectious amyloid fibers in vitro, enabling [SWI +] de novo formation through a simple transformation. Although the glutamine-rich middle region (Q) was not sufficient to aggregate in [SWI +] cells or essential for SWI/SNF function, it significantly modified the Swi1 aggregation pattern and Swi1 function. We also show that excessive Swi1 incurred Li+/Na+ sensitivity and that the N/Q regions are important for this gain of sensitivity. Taken together, our results provide the final proof of “protein-only” transmission of [SWI +] and demonstrate that the widely distributed “dispensable” glutamine/asparagine-rich regions/motifs might have important and divergent biological functions.

scholarly journals Structure of the rat platelet factor 4 gene: a marker for megakaryocyte differentiation.

1987 ◽  
Vol 7 (2) ◽  
pp. 898-904 ◽  
Author(s):  
T Doi ◽  
S M Greenberg ◽  
R D Rosenberg
Keyword(s):  
Amino Acid ◽  
Sequence Analysis ◽  
Genomic Library ◽  
Transcriptional Start Site ◽  
Platelet Factor 4 ◽  
Start Codon ◽  
Cdna Expression Library ◽  
Amino Acid Residues ◽  
Platelet Factor ◽  
Nuclease Mapping

A rat platelet factor 4 (PF4) cDNA has been isolated by immunoscreening a g lambda 11 rat megakaryocyte cDNA expression library. Sequence analysis of the rat PF4 cDNA revealed that this megakaryocyte protein is composed of a leader sequence of 29 amino acid residues and a mature protein sequence of 76 amino acid residues. The structure of rat PF4 derived from its cDNA shows a marked homology with the amino acid sequence of human PF4 obtained by classical protein chemistry techniques. This observation is particularly striking with regard to the carboxy-terminal region of rat and human PF4, where 28 of the last 31 C-terminal residues are identical. The rat PF4 gene was obtained from a rat genomic library by using rat PF4 cDNA as a hybridization probe. Sequence analysis showed that the gene is constructed of three exons and two short introns. The transcriptional start site is located 73 base pairs upstream of the translational start codon as judged by S1 nuclease mapping and primer extension. The 5' noncoding region of the gene also exhibited a sequence homologous to the TATA box at -31, as well as a series of direct and inverted repeat sequences and a cluster of 26 T residues at -155 to -218. This latter domain may be involved in regulating PF4 gene expression during megakaryocytopoiesis.

IDENTIFICATION, SEQUENCE AND EXPRESSION OF A CRUSTACEAN CARDIOACTIVE PEPTIDE (CCAP) GENE IN THE MOTHMANDUCA SEXTA

2001 ◽  
Vol 204 (16) ◽  
pp. 2803-2816 ◽  
Author(s):  
P. K. LOI ◽  
S. A. EMMAL ◽  
Y. PARK ◽  
N. J. TUBLITZ
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Gene Structure ◽  
Expression Patterns ◽  
Amino Acid Residues ◽  
Coding Region ◽  
Genome Database ◽  
Reading Frame ◽  
Crustacean Cardioactive Peptide ◽  
Embryonic Life

SUMMARYThe crustacean cardioactive peptide (CCAP) gene was isolated from the tobacco hawkmoth Manduca sexta. The gene has an open reading frame of 125 amino acid residues containing a single, complete copy of CCAP. Analysis of the gene structure revealed three introns interrupting the coding region. A comparison of the M. sexta CCAP gene with the Drosophila melanogaster genome database reveals significant similarities in sequence and gene structure.The spatial and temporal expression patterns of the CCAP gene in the M. sexta central nervous system were determined in all major post-embryonic stages using in situ hybridization techniques. The CCAP gene is expressed in a total of 116 neurons in the post-embryonic M. sextacentral nervous system. Nine pairs of cells are observed in the brain, 4.5 pairs in the subesophageal ganglion, three pairs in each thoracic ganglion(T1-T3), three pairs in the first abdominal ganglion (A1), five pairs each in the second to sixth abdominal ganglia (A2-A6) and 7.5 pairs in the terminal ganglion. The CCAP gene is expressed in every ganglion in each post-embryonic stage, except in the thoracic ganglia of first- and second-instar larvae. The number of cells expressing the CCAP gene varies during post-embryonic life,starting at 52 cells in the first instar and reaching a maximum of 116 shortly after pupation. One set of thoracic neurons expressing CCAP mRNA shows unusual variability in expression levels immediately prior to larval ecdysis. Using previously published CCAP immunocytochemical data, it was determined that 91 of 95 CCAP-immunopositive neurons in the M. sexta central nervous system also express the M. sexta CCAP gene, indicating that there is likely to be only a single CCAP gene in M. sexta.

A new species of the genus Tuberfemurus (Orthoptera: Tetrigoidea: Cladonotinae) with comments on the characters of mitochondrial genome

Zootaxa ◽  
2021 ◽  
Vol 5071 (3) ◽  
pp. 437-446
Author(s):  
MENG-QI WANG ◽  
YAO DENG ◽  
DE-LONG GUAN ◽  
BEN-YONG MAO ◽  
MIAO LI
Keyword(s):  
New Species ◽  
Mitochondrial Genome ◽  
Stop Codon ◽  
Complete Mitochondrial Genome ◽  
Hind Femur ◽  
Start Codon ◽  
Gene Arrangement ◽  
Coding Region ◽  
Phylogenetic Studies ◽  
A New Species

A new species, Tuberfemurus viridulus sp. nov. is described and illustrated with photographs. The new species is similar to T. torulisinotus Deng, 2019, but differs from the latter by broader vertex, invisible frontal costa in profile, distinctly truncate apex of hind pronotal process, and two large triangular projections on lower outer carinae of hind femur. An updated key to species of Tuberfemurus is provided. Simultaneously, the complete mitochondrial genome of Tuberfemurus viridulus sp. nov. is sequenced and analyzed. The total length of the assembled mitogenome is 15,060 bp with 37 typical mitochondrial genes and a non-coding region (A + T-rich region). The order and orientation of the gene arrangement pattern are identical to that of most Tetrigoidea species. All PCGs initiate with the standard start codon of ATN, except ATP6 with GAC and ND1 with TTG; and terminate with the complete stop codon (TAA/TAG) or with an incomplete T- codon. This data could provide the genome information available for Tetrigoidea and facilitate phylogenetic studies of related insects.  

scholarly journals Recovery of active beta-lactamases from Proteus vulgaris and RTEM-1 hybrid by random mutagenesis by using a dnaQ strain of Escherichia coli.

1996 ◽  
Vol 40 (9) ◽  
pp. 2152-2159 ◽  
Author(s):  
S M Hosseini-Mazinani ◽  
E Nakajima ◽  
Y Ihara ◽  
K Z Kameyama ◽  
K Sugimoto
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Random Mutagenesis ◽  
Amino Acid Residues ◽  
Proteus Vulgaris ◽  
Coding Region ◽  
Beta Lactamases ◽  
Functional Roles ◽  
Hybrid Genes ◽  
Lactamase Gene

Proteus vulgaris and RTEM-1 beta-lactamases that belong to molecular class A with 37% amino acid similarity were examined to find the relationship between amino acid residues and activity of enzymes. MICs of ampicillin were > 2,000 micrograms/ml for Escherichia coli cells producing these enzymes. We have made 18 hybrid genes by substituting the coding region of the P. vulgaris beta-lactamase gene with the equivalent portions from the RTEM-1 gene. Most of these hybrids produced inactive proteins, but a few hybrid enzymes had partial or trace activity. From one of the hybrid genes (MIC of ampicillin, 100 micrograms/ml), we recovered three kinds of active mutants which provided ampicillin MICs of 1,000 micrograms/ml by the selection of spontaneous mutations in a dnaQ strain of E. coli. In these mutants, Leu-148, Met-182, and Tyr-274 were replaced with Val, Thr, and His, respectively. These amino acids have not been identified as residues with functional roles in substrate hydrolysis. Furthermore, from these hybrid mutants, we obtained a second set of mutants which conferred ampicillin MICs of 1,500 micrograms/ml. Interestingly, the second mutations were limited to these three amino acid substitutions. These amino acid residues which do not directly interact with substrates have an effect on enzyme activity. These mutant enzymes exhibited lower K(m) values for cephaloridine than both parental enzymes.

scholarly journals The cDNA and protein sequences of human lactate dehydrogenase B

1987 ◽  
Vol 248 (3) ◽  
pp. 933-936 ◽  
Author(s):  
I Sakai ◽  
F S Sharief ◽  
Y C Pan ◽  
S S Li
Keyword(s):  
Amino Acid ◽  
Lactate Dehydrogenase ◽  
Amino Acid Composition ◽  
Acid Composition ◽  
Amino Acid Sequences ◽  
British Library ◽  
Amino Acid Residues ◽  
Coding Region ◽  
Cdna Insert ◽  
Protein Coding

Human lactate dehydrogenase B (LDH-B) cDNA was isolated and sequenced. The LDH-B cDNA insert consists of the protein-coding sequence (999 bp), the 5′ (54 bp) and 3′ (203 bp) non-coding regions, and the poly(A) tail (50 bp). The predicted sequence of 333 amino acid residues was confirmed by amino acid composition and/or sequence analyses of a total of 185 (56%) residues from tryptic peptides of human LDH-B protein. The nucleotide and amino acid sequences of the human LDH-B coding region show 68% and 75% homologies respectively with those of the human LDH-A. The peptide map and amino acid composition data have been deposited as Supplementary Publication SUP 50139 (7 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies are available on prepayment [see Biochem. J. (1987) 241, 5].

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