TY  - JOUR
A1  - Tinajero-Trejo, Mariana
A1  - Rana, Namrata
A1  - Nagel, Christoph
A1  - Jesse, Helen E.
A1  - Smith, Thomas W.
A1  - Wareham, Lauren K.
A1  - Hippler, Michael
A1  - Schatzschneider, Ulrich
A1  - Poole, Robert K.
T1  - Antimicrobial Activity of the Manganese Photoactivated Carbon Monoxide-Releasing Molecule [Mn(CO)\(_3\)(tpa-kappa\(^3\)N)]\(^+\) Against a Pathogenic Escherichia coli that Causes Urinary Infections
JF  - Antioxidants & Redox Signaling
N2  - Aims: We set out to investigate the antibacterial activity of a new Mn-based photoactivated carbon monoxide-releasing molecule (PhotoCORM, [Mn(CO)\(_3\)(tpa-kappa\(^3\)N)]\(^+\)) against an antibiotic-resistant uropathogenic strain (EC958) of Escherichia coli. Results: Activated PhotoCORM inhibits growth and decreases viability of E. coli EC958, but non-illuminated carbon monoxide-releasing molecule (CORM) is without effect. NADH-supported respiration rates are significantly decreased by activated PhotoCORM, mimicking the effect of dissolved CO gas. CO from the PhotoCORM binds to intracellular targets, namely respiratory oxidases in strain EC958 and a bacterial globin heterologously expressed in strain K-12. However, unlike previously characterized CORMs, the PhotoCORM is not significantly accumulated in cells, as deduced from the cellular manganese content. Activated PhotoCORM reacts avidly with hydrogen peroxide producing hydroxyl radicals; the observed peroxide-enhanced toxicity of the PhotoCORM is ameliorated by thiourea. The PhotoCORM also potentiates the effect of the antibiotic, doxycycline. Innovation: The present work investigates for the first time the antimicrobial activity of a light-activated PhotoCORM against an antibiotic-resistant pathogen. A comprehensive study of the effects of the PhotoCORM and its derivative molecules upon illumination is performed and mechanisms of toxicity of the activated PhotoCORM are investigated. Conclusion: The PhotoCORM allows a site-specific and time-controlled release of CO in bacterial cultures and has the potential to provide much needed information on the generality of CORM activities in biology. Understanding the mechanism(s) of activated PhotoCORM toxicity will be key in exploring the potential of this and similar compounds as antimicrobial agents, perhaps in combinatorial therapies with other agents.
KW  - intracellular hydrogen-peroxide
KW  - campylobacter-jejuni
KW  - oxygen-metabolism
KW  - deficient mutant
KW  - oxidative stress
KW  - aqueous-solution
KW  - metal caponyls
KW  - RU(CO)(3)CL(GLYCINATE)
KW  - bacteria
KW  - enzyme
Y1  - 2016
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-188910
VL  - 24
IS  - 14
ER  - 
TY  - THES
A1  - Merdanovic, Melisa
T1  - Charakterisierung von NadR : das essentielle Enzym der NAD-Synthese bei Haemophilus influenzae
T1  - Characterization of NadR: an essential enzyme of NAD synthesis in Haemophilus influenzae
N2  - I Zusammenfassung Haemophilus influenzae, ein Gram-negatives, Bakterium der Familie Pasteurellaceae, kann beim Menschen eine Vielzahl an Erkrankungen auslösen: Die bekapselte Stämme, v. a. mit Typ b Kapsel können Cellulitis, septische Arthritis, Epiglottitis und Meningitis verursachen. Die nicht-bekapselte Stämme können Otitis media, Sinusitis, Pneumonie und in selteneren Fällen Bakterämie verursachen. Ein besonderes Merkmal des Metabolismus von H. influenzae ist dessen Unfähigkeit Nikotinamid-Adenin-Dinukleotid (NAD+) de novo zu synthetisieren. Daher sind die Enzyme bzw. Transporter, die an NAD+ Aufnahme und Resynthese beteiligt sind, als putative antimikrobielle Ziele von Interesse. In unserer Arbeitsgruppe konnte gezeigt werden, dass NAD+ zu Nikotinamidribosyl degradiert werden muss, bevor es in die Zelle aufgenommen werden kann. Auch Proteine, die an der Degradation des exogenen NAD+ zu Nikotinamidribosyl und dessen anschließender Aufnahme in die Zelle verantwortlich sind, konnten identifiziert und charakterisiert werden. Wie Nikotinamidribosyl im Cytoplasma wiederum zu NAD+ synthetisiert wird, ist auch erst kürzlich geklärt worden: für NadR konnte sowohl eine Ribosyl-Nukleotid-Kinase (RNK) Aktivität als auch eine Nikotinamid-Mononukleotid-Adenylyltransferase (NMNAT) Aktivität in vitro gezeigt werden. Die Kristallstruktur von hiNadR im Komplex mit NAD+ wurde auch aufgeklärt. In dieser Arbeit sollte NadR, insbesondere dessen RNK Domäne, in vivo und in vitro näher charakterisiert werden. Um zu untersuchen, ob beide Domänen in vivo essentiell sind, wurden Deletionsmutanten erzeugt, bei welchen die komplette bzw. der C-terminale Teil der RNK Domäne fehlten. Diese Deletionen konnten im nadV+ Hintergrund erzeugt werden. Die Deletionen konnten in H. influenzae nur zusammen mit dem nadV-Gen transferiert werden oder alternativ nur in die Zellen, die mit pNadRKan Plasmid transformiert wurden. Dies verdeutlicht, dass nicht nur die NMNAT Domäne sondern auch die RNK Domäne bzw. sogar nur wenige C-terminal fehlende Aminosäuren des NadR Proteins essentiell für die Lebensfähigkeit von H. influenzae sind. Gleichzeitig zeigen diese Experimente, dass die RNK-Domäne in Anwesenheit von NadV redundant ist. Ein weiterer Phänotyp der RNK-Deletionsmutante zeigte sich beim Nikotinamidribosyl-Transport. Im Gegensatz zum Wt, welcher ca. 60-80% des 14C-Nikotinamidribosyls aufnahm, konnte für die RNK-Deletionsmutante nur 2-5% Aufnahme gemessen werden. Dies konnte durch das pNadRKan Plasmid komplementiert werden. Weiterhin wurde festgestellt, dass spontan Aminopyridin-resistente H. influenzae Zellen Mutationen im nadR Gen haben, insbesondere im Walker A-Motif (P-Loop) der RNK Domäne. Zusätzlich konnte in dieser Arbeit gezeigt werden, dass NadR aus Aminopyridin und ATP Aminopyridin-Adenin-Dinukleotid synthetisieren kann. Somit konnte gezeigt werden, dass die wachstumshemmende Wirkung eigentlich durch das aus Aminopyridin synthetisierte Aminopyridin-Adenin-Dinukleotid entsteht, welches NAD+ in Redox-Reaktionen verdrängt, wodurch es letztendlich zum Stillstand des Metabolimus kommt. Durch Einführen von gezielten AS-Substitutionen im Walker A und B Motif und in der LID-Domäne von NadR, konnten einige Aminosäuren identifiziert werden, welche essentiell für die Aktivität der RNK Domäne sind. Alle Aminosäuren-Substitutionen führten zum Verlust der RNK Aktivität, die NMNAT Aktivität jedoch war nicht beeinträchtigt. Desweiteren wurden diese NadR Punktmutanten in vivo untersucht. Für alle konnte eine signifikante Defizienz in der Nikotinamidribosyl-Aufnahme beobachtet werden, die gemessene Aufnahme lag im Bereich der RNK-Deletionsmutante. Dadurch konnte eine direkte Korrelation zwischen der RNK Aktivität und der Nikotinamidribosyl-Aufnahme gezeigt werden. In weiteren in vitro Experimenten konnte für NadR eine Feedback-Inhibition durch das NAD+ gezeigt werden, wobei NAD+ in erster Linie die RNK Domäne von NadR inhibiert. Eine graduelle Erhöhung der NAD+ Konzentration führte in den in vitro Assays zu einer graduellen Abnahme der RNK. Bei der NMNAT Aktivität jedoch zeigte sich keine signifikante Inhibition in Anwesenheit von NAD+. Begleitende in vivo Experimente, zeigten eine 2/3 Reduktion der Nikotinamidribosyl-Aufnahme bei den Zellen, die mit NAD+ inkubiert wurden, d. h. höhere intrazelluläre NAD+ Konzentration hatten. Für die genauere Analyse der Feedback-Inhibition durch NAD+ wurden weitere Punktmutanen hergestellt. Bei zwei der Punktmutanten wurde eine Beeinträchtigung der NadR-Aktivität beobachtet, daher wurden diese Punktmutanten von weiteren Analysen im Bezug auf NAD+-Feedback Inhibition ausgeschlossen. Eine Mutante (NadRW256F) jedoch, zeigte ähnliche Aktivität wie das Wt-NadR. In Anwesenheit von NAD+ wurde die RNK Aktivität dieser Punktmutante, im Gegensatz zum Wt-Protein, kaum gehemmt. Dadurch konnte W256 als eine der Aminosäuren identifiziert werden, die an der Vermittlung der NAD+-bedingten Inhibition der RNK-Domäne beteiligt ist.
N2  - I Summary Haemophilus influenzae, a gram-negative human pathogen belonging to a family of Pasteurellaceae is a causative agent of several distinct diseases. Whereas capsulated strains, particulary those with tybe b capsule can cause severe invasive infections such as cellulitis, septic arthrithis, epiglottitis and meningitis, non-capsulated strains generally tend to cause localized disease including otitis media, sinusitis, pneumonia and in rare cases bacteremia. The inability to synthesize NAD+ de novo is one of the hallmarks of H. influenzae metabolism, therefore proteins involved in NAD+ uptake and utilization respresent interesting putative targets for development of novel antimicrobial treatment. In our lab we were able to show, that prior to uptake, NAD+ has to be degraded to NR. Several proteins involved in NAD+ degradation and NR uptake were identified and characterized: OmpP2 (an outer-membrane porin), e(P4) (a membrane-bound acid phosphoesterase), NadN (a periplasmatic nucleotidase) and PnuC (a nicotinamidribosyl transporter localized in inner membrane). Enzyme responsible for resynthesis of nicotinamidribosyl to NAD+ was recently found to be NadR: A bifunctional protein containing a nicotinamidribosyl kinase (RNK) and a nicotinamid mononucleotide adenylyltransferase (NMNAT) activity, both of which were confirmed in vitro. Also, the crystal structure of NadR complexed with NAD+ was recently resolved. The aim of this work was to characterize the in vivo function of NadR, particular interest was laid on the characterization of the nicotinamidribosyl kinase domain. To test if both domains of NadR are essential for survival, deletion mutants lacking the entire RNK domain and the C-terminal 58 amino acids were constructed. Initially, these mutants were made in a H. influenzae strain which contains a chromosomal copy of H. ducreyi nadV gene. In following transformation experiments, transfer of the RNK deletion mutants to H. influenzae strain was always accompanied with an nadV transfer as well. Only in strain containing pNadRKan plasmid, no nadV transfer along with RNK-deletions took place. Indirectly, this shows that not only the entire RNK domain is essential for H. influenzae, but also the last 58 amino acids as well. It also shows that in presence of NadV the RNK domain is redundant. RNK deletion mutant displayed a significant deficiency in nicotinamidribosyl transport as well: whereas the Wt strain can accumulate up to 80% of 14C labeled nicotinamidribosyl, RNK mutant is able to accumulate only 2-5%. Introduction of pNadRKan plasmid to RNK mutant restored transport efficiency to Wt level. Studies using spontanous 3-aminopyridine (3-AmPR) resistant H. influenzae isolates, revealed that almost all 3-AmPR resistant isolates have mutations in the nadR gene. A clustering of mutations in Walker A motif of the RNK domain could be observed. Further studies represented in this work, show that 3-AmPR can act as a subtrate for NadR, therefore in ATP consuming reactions aminopyridine-adenindinucleotide can be synthesized. Intracellular aminopyridine-adenindinucleotide replaces NAD+ in redox reactions, which ultimately leads to inhibition of cell metabolism, thereby explaining the mechanism of 3-AmPR based growth inhibition. Using site-directed mutagenesis to introduce amino acid substitutions in distinct parts of the NadR-RNK domain, active sites of the RNK domain were revealed and amino acids essential for the RNK activity were identified. These defined amino acid exchanges resulted in loss of the RNK activity in vitro, but had no effect on the NMNAT activity, which remained intact in these mutant variants of NadR. Following in vivo studies revealed that all mutant NadR proteins caused a severe nicotinamidribosyl uptake deficiency, similar to the one observed in the RNK deletion mutant. Therefore, a direct correlation between the RNK activity and nicotinamidribosyl uptake was shown. Further in vitro studies revealed a feedback inhibition of NadR by NAD+, especially for the RNK domain. In case of RNK domain a gradual increase of NAD+ concentration led to gradual decrease in RNK activity. In contrast, for NMNAT domain no significant inhibition in the presence of NAD+ was observed. Also, in in vivo experiments a 3 fold reduction of nicotinamidribosyl uptake rate was observed when intracellular NAD+ concentrations were higher. To adress the mechanism of NAD+ feedback inhibition, once again, distinct amino acid exchanges were introduced. In vitro, two of the mutant proteins were impaired in their activity, especially if lower protein contrations were used. Therefore, further test concerning inhibtion were not preformed with these mutants. However, a W256F protein displayed activity similar to that of the native protein and furthermore was not inhibited in presence of NAD+. This indicates an involvement of the amino acid W256 in mediating the NAD+ dependent feedback inhibition on NadR activity.
KW  - Haemophilus influenzae
KW  - NAD
KW  - Synthese
KW  - NAD
KW  - Enzym
KW  - Synthese
KW  - Haemophilus
KW  - influenzae
KW  - NAD
KW  - enzyme
KW  - synthesis
KW  - Haemophilus
KW  - influenzae
Y1  - 2005
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-14907
ER  - 
TY  - JOUR
A1  - Scherer, Marc
A1  - Fleishman, Sarel J.
A1  - Jones, Patrik R.
A1  - Dandekar, Thomas
A1  - Bencurova, Elena
T1  - Computational Enzyme Engineering Pipelines for Optimized Production of Renewable Chemicals
JF  - Frontiers in Bioengineering and Biotechnology
N2  - To enable a sustainable supply of chemicals, novel biotechnological solutions are required that replace the reliance on fossil resources. One potential solution is to utilize tailored biosynthetic modules for the metabolic conversion of CO2 or organic waste to chemicals and fuel by microorganisms. Currently, it is challenging to commercialize biotechnological processes for renewable chemical biomanufacturing because of a lack of highly active and specific biocatalysts. As experimental methods to engineer biocatalysts are time- and cost-intensive, it is important to establish efficient and reliable computational tools that can speed up the identification or optimization of selective, highly active, and stable enzyme variants for utilization in the biotechnological industry. Here, we review and suggest combinations of effective state-of-the-art software and online tools available for computational enzyme engineering pipelines to optimize metabolic pathways for the biosynthesis of renewable chemicals. Using examples relevant for biotechnology, we explain the underlying principles of enzyme engineering and design and illuminate future directions for automated optimization of biocatalysts for the assembly of synthetic metabolic pathways.
KW  - computational
KW  - enzyme
KW  - engineering
KW  - design
KW  - biomanufacturing
KW  - biofuel
KW  - microbes
KW  - metabolism
Y1  - 2021
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-240598
SN  - 2296-4185
VL  - 9
ER  - 
TY  - JOUR
A1  - Noll, Niklas
A1  - Krause, Ana-Maria
A1  - Beuerle, Florian
A1  - Würthner, Frank
T1  - Enzyme-like water preorganization in a synthetic molecular cleft for homogeneous water oxidation catalysis
JF  - Nature Catalysis
N2  - Inspired by the proficiency of natural enzymes, mimicking of nanoenvironments for precise substrate preorganisation is a promising strategy in catalyst design. However, artificial examples of enzyme-like activation of H\(_2\)O molecules for the challenging oxidative water splitting reaction are hardly explored. Here, we introduce a mononuclear Ru(bda) complex (M1, bda: 2,2’-bipyridine-6,6’-dicarboxylate) equipped with a bipyridine-functionalized ligand to preorganize H\(_2\)O molecules in front of the metal center as in enzymatic clefts. The confined pocket of M1 accelerates chemically driven water oxidation at pH 1 by facilitating a water nucleophilic attack pathway with a remarkable turnover frequency of 140 s\(^{−1}\) that is comparable to the oxygen-evolving complex of photosystem II. Single crystal X-ray analysis of M1 under catalytic conditions allowed the observation of a 7th H\(_2\)O ligand directly coordinated to a RuIII center. Via a well-defined hydrogen-bonding network, another H\(_2\)O substrate is preorganized for the crucial O–O bond formation via nucleophilic attack.
KW  - water oxidation
KW  - enzyme
KW  - catalysis
KW  - molecular
KW  - catalyst synthesis
KW  - catalytic mechanisms
KW  - homogeneous catalysis
KW  - photocatalysis
KW  - supramolecular chemistry
Y1  - 2022
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-302897
N1  - This version of the article has been accepted for publication, after peer review and is subject to Springer Nature’s AM terms of use (https://www.springernature.com/gp/open-research/policies/accepted-manuscript-terms), but is not the Version of Record and does not reflect post-acceptance improvements, or any corrections. The Version of Record is available online at: https://doi.org/10.1038/s41929-022-00843-x
ET  - accepted version
ER  - 
TY  - THES
A1  - Albers, Christine
T1  - Reinigung und Charakterisierung der alpha-Methylacyl-CoA-Racemase aus menschlicher Leber
T1  - Purification and characterisation of alpha-Methylacyl-CoA-Racemase from human liver
N2  - Im Katabolismus methylverzweigter Fettsäuren spielt die alpha-Methylacyl-CoA-Racemase eine wichtige Rolle, indem sie die (R)- und (S)-Isomere von alpha-methylverzweigten Fettsäuren als Coenzym A Thioester racemisiert. Methylverzweigte Fettsäuren entstehen beim Abbau von Isoprenoiden und werden darüber hinaus auch von vielen Organismen, wie z.B. Mycobakterien, synthetisiert. Die Hauptaufgabe der Racemase ist aber vermutlich in der Biosynthese von Gallensäuren zu sehen. Das Ziel der vorliegenden Arbeit war es, die alpha-Methylacyl-CoA-Racemase aus humanem Gewebe zu reinigen und zu charakterisieren sowie ihre physiologische Rolle im Katabolismus verzweigtkettiger Fettsäuren und der Gallensäurebiosynthese zu untersuchen. Die alpha-Methylacyl-CoA-Racemase wurde aus humanem Gewebe zur Homogenität gereinigt, umfassend biochemisch charakterisiert und zur genauen molekularbiologischen Analyse in E.coli kloniert. Die Aktivität der Racemase wurde anhand der [³H]H2O-Freisetzung aus [alpha-³H]-a-Methylacyl-CoAs bestimmt. Die humane Racemase ist in der aktiven Form ein monomeres Protein und besteht aus 382 Aminosäuren. Als Substrate akzeptiert das Enzym ein breites Spektrum von alpha-Methylacyl-CoAs. Neben den Coenzym A-Thioestern alpha-methylverzweigter Fettsäuren, wie Pristansäure, werden auch CoA-Ester von Steroidderivaten, z.B. des Gallensäureintermediats Trihydroxycoprostansäure, und aromatischen Phenylpropionsäuren, wie dem Analgetikum Ibuprofen, umgesetzt. Freie Fettsäuren, geradkettige oder beta-methylverzweigte Acyl-CoAs werden nicht racemisiert. Die alpha-Methylacyl-CoA-Racemase ist im Menschen zu ca. 80 Prozent auf die Peroxisomen und ca. 20 Prozent auf die Mitochondrien verteilt, wobei entsprechende peroxisomale (PTS 1) und mitochondriale (MTS) Transportsignale die Lokalisation bestimmen. Die vollständige cDNA-Sequenz der humanen a-Methylacyl-CoA-Racemase hat eine Gesamtlänge von 2039 Basenpaaren mit einem offenen Leseraster von 89 - 1237 bp. Das Startcodon ATG ist in eine klassische Kozak-Sequenz zum Translationsstart eingebettet. Die Protein endet am C-Terminus mit dem Sequenzmotiv –KASL, das dem peroxisomalen Transportsignal (PTS I) einiger Säugetierkatalasen entspricht. Aufgrund alternativer Polyadenylierung sind in allen untersuchten menschlichen Geweben Transkripte von 1,6 kb bzw. 2,0 kb zu finden. Es liegt keine gewebsabhängige Polyadenylierung vor, die Racemase wird aber gewebsspezifisch exprimiert (besonders stark in Leber und Niere). Das humane Racemasegen liegt auf dem kurzen Arm des Chromosoms 5 nahe am Centromer (5p1.3), im Intervall von D5S651 (46,6 cM) und D5S634 (59.9 cM).
N2  - Racemization is an essential step for bile acid synthesis and it is important for degradation of alpha-methyl branched-chain fatty acids. The (R)- and (S)-isomers of alpha-methyl-branched chain fatty acids were shown to be interconverted as coenzyme A thioesters by an alpha-methylacyl-CoA racemase. Various branched-chain fatty acids arise in the catabolism of isoprenoids and are also synthesized by a variety of organisms, particularly mycobacteria. The aim of this work was to purify and to characterize the racemase from human tissue and to analyse the physiological role in the degradation of branched-chain fatty acids and the bile acid synthesis. The alpha-methylacyl-CoA racemase was purified from human liver to apparent homogeneity. The enzyme was exhaustively characterized by methods of biochemistry and protein chemistry. The cDNA coding for human racemase was cloned in E. coli and sequenced. A radiometric assay with 2-methyl[2-³H]acyl-CoAs as substrates was used routinely for monitoring purification procedure. The active form of the enzyme is a monomeric protein comprising 382 amino acids. The enzyme accepts a wide range of alpha-methylacyl-CoAs, including pristanoyl-CoA, trihydroxycoprostanoyl-CoA (an intermediate in bile acid synthesis) as substrates. Also arylpropionyl-CoAs such as the anti-inflammatory drug ibuprofen are accepted, but neither free fatty acids, beta-methyl-branched nor linear-chain acyl-CoAs. In human tissues 80 - 90 Prozent of the racemase activity is found in peroxisomes and 10 - 20 Prozent in mitochondria. Degradation of branched chain fatty acids is located in both compartments, so the enzyme has to be distributed between peroxisomes and mitochondria. No evidence was found for the existence of isoenzymes or different transcription products. It appears that only one mRNA is transcribed from one gene and that also only one protein is synthesized. The different recognition of peroxisomal (PTS 1) and mitochondrial targeting signals (MTS) may determine the subcellular distribution. The complete cDNA sequence has an overall length of 2039 base pairs, with a open reading frame between 89 - 1237 bp. The ATG start codon is embedded in a classical Kozak sequence for translation start. The C-Terminus of the protein is –KASL, which is very similar to the peroxisomal targeting signals (PTS 1) of many mammalian catalases. In all human tissues analysed in this work two different transcripts of racemase with sizes of 1,6 kb and 2,0 kb have been found and show alternate polyadenylation. Polyadenylation of racemase is not tissue-dependent but its expression is tissue-specific (strong activity is found in liver and kidney). The human racemase gene is localized on the short arm of chromosome 5, near the centromer (region 5p1.3) and between the markers D5S651 (46,6 cM) and D5S634 (59.9 cM).
KW  - Alpha-Methylacyl-CoA racemase
KW  - Mensch
KW  - Leber
KW  - Molekularbiologie
KW  - Racemase
KW  - human
KW  - Enzym
KW  - Reinigung
KW  - Charakterisierung
KW  - Peroxisom
KW  - alpha-Methylacyl-CoA
KW  - Racemase
KW  - human
KW  - enzyme
KW  - purification
KW  - characterisation
KW  - peroxisome
KW  - alpha-Methylacyl-CoA
Y1  - 2000
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-770
ER  - 
TY  - JOUR
A1  - Krehan, Mario
A1  - Heubeck, Christian
A1  - Menzel, Nicolas
A1  - Seibel, Peter
A1  - Schön, Astrid
T1  - RNase MRP RNA and RNase P activity in plants are associated with a Pop1p containing complex
JF  - Nucleic Acids Research
N2  - RNase P processes the 5'-end of tRNAs. An essential catalytic RNA has been demonstrated in Bacteria, Archaea and the nuclei of most eukaryotes; an organism-specific number of proteins complement the holoenzyme. Nuclear RNase P from yeast and humans is well understood and contains an RNA, similar to the sister enzyme RNase MRP. In contrast, no protein subunits have yet been identified in the plant enzymes, and the presence of a nucleic acid in RNase P is still enigmatic. We have thus set out to identify and characterize the subunits of these enzymes in two plant model systems. Expression of the two known Arabidopsis MRP RNA genes in vivo was verified. The first wheat MRP RNA sequences are presented, leading to improved structure models for plant MRP RNAs. A novel mRNA encoding the central RNase P/MRP protein Pop1p was identified in Arabidopsis, suggesting the expression of distinct protein variants from this gene in vivo. Pop1p-specific antibodies precipitate RNase P activity and MRP RNAs from wheat extracts. Our results provide evidence that in plants, Pop1p is associated with MRP RNAs and with the catalytic subunit of RNase P, either separately or in a single large complex.
KW  - enzyme
KW  - binding
KW  - sequence
KW  - cyanelle
KW  - in vitro
KW  - partial purification
KW  - protein subunit
KW  - ribonuclease-P
KW  - genes
KW  - identification
Y1  - 2012
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130648
VL  - 40
IS  - 16
ER  -