Pyridoxamine-phosphate oxidases and pyridoxamine-phosphate oxidase-related proteins catalyze the oxidation of 6-NAD(P)H to NAD(P)+

2019 ◽  
Vol 476 (20) ◽  
pp. 3033-3052 ◽  
Author(s):  
Alexandre Y. Marbaix ◽  
Georges Chehade ◽  
Gaëtane Noël ◽  
Pierre Morsomme ◽  
Didier Vertommen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Arabidopsis Thaliana ◽  
Mammalian Cells ◽  
Reduced Form ◽  
Nostoc Punctiforme ◽  
Catalytic Function ◽  
Related Proteins ◽  
Glucose 6 Phosphate Dehydrogenase

Abstract 6-NADH and 6-NADPH are strong inhibitors of several dehydrogenases that may form spontaneously from NAD(P)H. They are known to be oxidized to NAD(P)+ by mammalian renalase, an FAD-linked enzyme mainly present in heart and kidney, and by related bacterial enzymes. We partially purified an enzyme oxidizing 6-NADPH from rat liver, and, surprisingly, identified it as pyridoxamine-phosphate oxidase (PNPO). This was confirmed by the finding that recombinant mouse PNPO oxidized 6-NADH and 6-NADPH with catalytic efficiencies comparable to those observed with pyridoxine- and pyridoxamine-5′-phosphate. PNPOs from Escherichia coli, Saccharomyces cerevisiae and Arabidopsis thaliana also displayed 6-NAD(P)H oxidase activity, indicating that this ‘side-activity’ is conserved. Remarkably, ‘pyridoxamine-phosphate oxidase-related proteins’ (PNPO-RP) from Nostoc punctiforme, A. thaliana and the yeast S. cerevisiae (Ygr017w) were not detectably active on pyridox(am)ine-5′-P, but oxidized 6-NADH, 6-NADPH and 2-NADH suggesting that this may be their main catalytic function. Their specificity profiles were therefore similar to that of renalase. Inactivation of renalase and of PNPO in mammalian cells and of Ygr017w in yeasts led to the accumulation of a reduced form of 6-NADH, tentatively identified as 4,5,6-NADH3, which can also be produced in vitro by reduction of 6-NADH by glyceraldehyde-3-phosphate dehydrogenase or glucose-6-phosphate dehydrogenase. As 4,5,6-NADH3 is not a substrate for renalase, PNPO or PNPO-RP, its accumulation presumably reflects the block in the oxidation of 6-NADH. These findings indicate that two different classes of enzymes using either FAD (renalase) or FMN (PNPOs and PNPO-RPs) as a cofactor play an as yet unsuspected role in removing damaged forms of NAD(P).

scholarly journals Cells Expressing Murine RAD52 Splice Variants Favor Sister Chromatid Repair

2006 ◽  
Vol 26 (10) ◽  
pp. 3752-3763 ◽  
Author(s):  
Peter H. Thorpe ◽  
Vanessa A. Marrero ◽  
Margaret H. Savitzky ◽  
Ivana Sunjevaric ◽  
Tom C. Freeman ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Sister Chromatid ◽  
Mammalian Cells ◽  
Splice Variants ◽  
Protein A ◽  
Single Stranded Dna ◽  
Culture Cells ◽  
Dominant Phenotype ◽  
Mouse Tissues

ABSTRACT The RAD52 gene is essential for homologous recombination in the yeast Saccharomyces cerevisiae. RAD52 is the archetype in an epistasis group of genes essential for DNA damage repair. By catalyzing the replacement of replication protein A with Rad51 on single-stranded DNA, Rad52 likely promotes strand invasion of a double-stranded DNA molecule by single-stranded DNA. Although the sequence and in vitro functions of mammalian RAD52 are conserved with those of yeast, one difference is the presence of introns and consequent splicing of the mammalian RAD52 pre-mRNA. We identified two novel splice variants from the RAD52 gene that are expressed in adult mouse tissues. Expression of these splice variants in tissue culture cells elevates the frequency of recombination that uses a sister chromatid template. To characterize this dominant phenotype further, the RAD52 gene from the yeast Saccharomyces cerevisiae was truncated to model the mammalian splice variants. The same dominant sister chromatid recombination phenotype seen in mammalian cells was also observed in yeast. Furthermore, repair from a homologous chromatid is reduced in yeast, implying that the choice of alternative repair pathways may be controlled by these variants. In addition, a dominant DNA repair defect induced by one of the variants in yeast is suppressed by overexpression of RAD51, suggesting that the Rad51-Rad52 interaction is impaired.

Functional interaction between p21rap1A and components of the budding pathway in Saccharomyces cerevisiae

1992 ◽  
Vol 12 (9) ◽  
pp. 4084-4092
Author(s):  
P C McCabe ◽  
H Haubruck ◽  
P Polakis ◽  
F McCormick ◽  
M A Innis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Bound States ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Amino Terminal ◽  
Loop Region

The rap1A gene encodes a 21-kDa, ras-related GTP-binding protein (p21rap1A) of unknown function. A close structural homolog of p21rap1A (65% identity in the amino-terminal two-thirds) is the RSR1 gene product (Rsr1p) of Saccharomyces cerevisiae. Although Rsr1p is not essential for growth, its presence is required for nonrandom selection of bud sites. To assess the similarity of these proteins at the functional level, wild-type and mutant forms of p21rap1A were tested for complementation of activities known to be fulfilled by Rsr1p. Expression of p21rap1A, like multicopy expression of RSR1, suppressed the conditional lethality of a temperature-sensitive cdc24 mutation. Point mutations predicted to affect the localization of p21rap1A or its ability to cycle between GDP and GTP-bound states disrupted suppression of cdc24ts, while other mutations in the 61-65 loop region improved suppression. Expression of p21rap1A could not, however, suppress the random budding phenotype of rsr1 cells. p21rap1A also apparently interfered with the normal activity of Rsrlp, causing random budding in diploid wild-type cells, suggesting an inability of p21rap1A to interact appropriately with Rsr1p regulatory proteins. Consistent with this hypothesis, we found an Rsr1p-specific GTPase-activating protein (GAP) activity in yeast membranes which was not active toward p21rap1A, indicating that p21rap1A may be predominantly GTP bound in yeast cells. Coexpression of human Rap1-specific GAP suppressed the random budding due to expression of p21rap1A or its derivatives, including Rap1AVal-12. Although Rap1-specific GAP stimulated the GTPase of Rsr1p in vitro, it did not dominantly interfere with Rsr1p function in vivo. A chimera consisting of Rap1A1-165::Rsr1p166-272 did not exhibit normal Rsr1p function in the budding pathway. These results indicated that p21rap1A and Rsr1p share at least partial functional homology, which may have implications for p21rap1A function in mammalian cells.

scholarly journals Minimal and RNA-free RNase P in Aquifex aeolicus

2017 ◽  
Vol 114 (42) ◽  
pp. 11121-11126 ◽  
Author(s):  
Astrid I. Nickel ◽  
Nadine B. Wäber ◽  
Markus Gößringer ◽  
Marcus Lechner ◽  
Uwe Linne ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Rnase P ◽  
Aquifex Aeolicus ◽  
Trna Processing ◽  
Hyperthermophilic Bacterium ◽  
Domains Of Life

RNase P is an essential tRNA-processing enzyme in all domains of life. We identified an unknown type of protein-only RNase P in the hyperthermophilic bacterium Aquifex aeolicus: Without an RNA subunit and the smallest of its kind, the 23-kDa polypeptide comprises a metallonuclease domain only. The protein has RNase P activity in vitro and rescued the growth of Escherichia coli and Saccharomyces cerevisiae strains with inactivations of their more complex and larger endogenous ribonucleoprotein RNase P. Homologs of Aquifex RNase P (HARP) were identified in many Archaea and some Bacteria, of which all Archaea and most Bacteria also encode an RNA-based RNase P; activity of both RNase P forms from the same bacterium or archaeon could be verified in two selected cases. Bioinformatic analyses suggest that A. aeolicus and related Aquificaceae likely acquired HARP by horizontal gene transfer from an archaeon.

scholarly journals Potential probiotic yeast isolated from an Indonesian indigenous fermented fish (Ikan Budu)

10.5219/1544 ◽  
2021 ◽  
Vol 15 ◽  
pp. 460-466
Author(s):  
Yetti Marlida ◽  
Nurul Huda ◽  
Harnentis ◽  
Yuliaty Shafan Nur ◽  
Nuri Mekar Lestari ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Staphylococcus Aureus ◽  
Bile Acid ◽  
Salmonella Enteritidis ◽  
Pathogenic Bacteria ◽  
Acid Tolerance ◽  
Fermented Food ◽  
Probiotic Properties

Budu is a fermented food resulting from the activities of microorganisms like lactic acid bacteria and yeast. Budu, therefore, serves as a source of probiotics that can have beneficial effects on livestock and humans. Nonetheless, their selection has to be done with caution. The current study purposed to find out whether budu has desirable probiotic properties. This was done by determining its pH, bile acid tolerance, hydrophobicity, and inhibition of pathogens such as Staphylococcus aureus, Salmonella enteritidis, and Escherichia coli. An in vitro experiment was conducted using three Saccharomyces cerevisiae (coded as SC 11, SC 12, and SC 21) in the preparation of budu. The whole experiment was repeated four times. The budus were tested for their probiotic properties (low pH, bile salts, hydrophobicity, and inhibition of pathogenic bacteria). The results showed that the three Saccharomyces cerevisiae survived in gastric juice and bile acid, exhibited good hydrophobicity, and could inhibit pathogenic bacteria, both gram-positive and negative pathogens. They were able to survive at pH 2 for 3 h (40.70 to 55.1%), at pH 2 for 5 h (35.25 to 46.88%), in 0.3% bile acid incubated for 3 h (69.69 to 86.56%), in 0.3% bile acid incubated for 5 h (82.22 to 88.18%) and hydrophobicity ability of 97.0 to 98.1%. The inhibition activity against pathogenic bacteria, that is, Escherichia coli was 2.50 to 3.81 mm, Staphylococcus aureus was 1.66 to 3.71 mm, and Salmonella enteritidis was 1.20 to 2.64 mm.

scholarly journals Lactobacillus rhamnosus GG and Saccharomyces cerevisiae boulardii exert synergistic antipathogenic activity in vitro against enterotoxigenic Escherichia coli

2019 ◽  
Vol 10 (8) ◽  
pp. 923-935 ◽  
Author(s):  
F. Moens ◽  
C. Duysburgh ◽  
P. van den Abbeele ◽  
M. Morera ◽  
M. Marzorati
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Synergistic Effects ◽  
Lactobacillus Rhamnosus ◽  
Proximal Colon ◽  
Enterotoxigenic Escherichia Coli ◽  
Short Term ◽  
Lactobacillus Rhamnosus Gg ◽  
Mucosal Layer

Short-term colonic in vitro batch incubations were performed to elucidate the possible synergistic effects of Lactobacillus rhamnosus GG (CNCM-I-4798) and Saccharomyces cerevisiae boulardii (CNCM-I-1079) (associated in Smebiocta/Smectaflora Protect®) on the colonic microbial fermentation process, as well as their antipathogenic activity against enterotoxigenic Escherichia coli (LMG2092) (ETEC). These incubations adequately simulate the native microbiota and environmental conditions of the proximal colon of both adult and toddler donors, including the colonic mucosal layer. Results indicated that both strains were capable of growing together without showing antagonistic effects. Co-cultivation of both strains resulted in increased butyrate (stimulated by L. rhamnosus GG), propionate (stimulated by S. boulardii), and ethanol (produced by S. boulardii) production compared to the control incubations, revealing the additive effect of both strains. After inoculation of ETEC under simulated dysbiotic conditions, a 40 and 46% reduction in the concentration of ETEC was observed upon addition of both strains during the experiments with the adult and toddler donor, respectively. Furthermore, ETEC toxin levels decreased upon S. boulardii inoculation, probably due to proteolytic activity of this strain, with a synergistic effect being observed upon co-cultivation of L. rhamnosus GG and S. boulardii resulting in a reduction of 57 and 46% for the adult and toddler donor, respectively. Altogether, the results suggest that both probiotics together may help microbiota functionality, in both adults and toddlers and under healthy or impaired conditions, which could be of great interest when the colonic microbiota is dysbiotic and therefore sensitive to pathogenic invasion such as during antibiotic treatment.

scholarly journals Regulation of Greatwall kinase during Xenopus oocyte maturation

2011 ◽  
Vol 22 (13) ◽  
pp. 2157-2164 ◽  
Author(s):  
Tomomi M. Yamamoto ◽  
Kristina Blake-Hodek ◽  
Byron C. Williams ◽  
Andrea L. Lewellyn ◽  
Michael L. Goldberg ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Mammalian Cells ◽  
Xenopus Oocytes ◽  
Kinase Activity ◽  
Wild Type ◽  
M Phase ◽  
Phosphatase 2A ◽  
Greatwall Kinase ◽  
Related Proteins

Greatwall kinase has been identified as a key element in M phase initiation and maintenance in Drosophila, Xenopus oocytes/eggs, and mammalian cells. In M phase, Greatwall phosphorylates endosulfine and related proteins that bind to and inhibit protein phosphatase 2A/B55, the principal phosphatase for Cdk-phosphorylated substrates. We show that Greatwall binds active PP2A/B55 in G2 phase oocytes but dissociates from it when progesterone-treated oocytes reach M phase. This dissociation does not require Greatwall kinase activity or phosphorylation at T748 in the presumptive T loop of the kinase. A mutant K71M Greatwall, also known as Scant in Drosophila, induces M phase in the absence of progesterone when expressed in oocytes, despite its reduced stability and elevated degradation by the proteasome. M phase induction by Scant Greatwall requires protein synthesis but is not associated with altered binding or release of PP2A/B55 as compared to wild-type Greatwall. However, in vitro studies with Greatwall proteins purified from interphase cells indicate that Scant, but not wild-type Greatwall, has low but detectable activity against endosulfine. These results demonstrate progesterone-dependent regulation of the PP2A/B55–Greatwall interaction during oocyte maturation and suggest that the cognate Scant Greatwall mutation has sufficient constitutive kinase activity to promote M phase in Xenopus oocytes.

scholarly journals Histone H3 K4 Demethylation during Activation and Attenuation of GAL1 Transcription in Saccharomyces cerevisiae

2007 ◽  
Vol 27 (22) ◽  
pp. 7856-7864 ◽  
Author(s):  
Kristin Ingvarsdottir ◽  
Chris Edwards ◽  
Min Gyu Lee ◽  
Jung Shin Lee ◽  
David C. Schultz ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Gene Activation ◽  
Histone H3 ◽  
Histone Lysine ◽  
Insight Into ◽  
Demethylation Activity

ABSTRACT In mammalian cells, histone lysine demethylation is carried out by two classes of enzymes, the LSD1/BHC110 class and the jumonji class. The enzymes of the jumonji class in the yeast Saccharomyces cerevisiae have recently also been shown to have lysine demethylation activity. Here we report that the protein encoded by YJR119c (termed KDM5), coding for one of five predicted jumonji domain proteins in yeast, specifically demethylates trimethylated histone H3 lysine 4 (H3K4me3), H3K4me2, and H3K4me1 in vitro. We found that loss of KDM5 increased mono-, di-, and trimethylation of lysine 4 during activation of the GAL1 gene. Interestingly, cells deleted of KDM5 also displayed a delayed reduction of K4me3 upon reestablishment of GAL1 repression. These results indicate that K4 demethylation has two roles at GAL1, first to establish appropriate levels of K4 methylation during gene activation and second to remove K4 trimethylation during the attenuation phase of transcription. Thus, analysis of lysine demethylation in yeast provides new insight into the physiological roles of jumonji demethylase enzymes.

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