MGDG, PG and SQDG regulate the activity of light-dependent protochlorophyllide oxidoreductase

2017 ◽  
Vol 474 (7) ◽  
pp. 1307-1320 ◽  
Author(s):  
Michal Gabruk ◽  
Beata Mysliwa-Kurdziel ◽  
Jerzy Kruk
Keyword(s):  
Molecular Mechanisms ◽  
Chlorophyll Biosynthesis ◽  
Fluorescence Emission ◽  
Lipid Binding ◽  
Site Directed Mutagenesis ◽  
Biosynthesis Pathway ◽  
Amino Acid Residues ◽  
Protochlorophyllide Oxidoreductase ◽  
Emission Maximum ◽  
Identify Amino Acid

Light-dependent protochlorophyllide oxidoreductase (POR) is a plant enzyme involved in the chlorophyll biosynthesis pathway. POR reduces one of the double bonds of the protochlorophyllide (Pchlide) using NADPH and light. In the present study, we found out that phosphatidylglycerol and sulfoquinovosyl diacylglycerol are allosteric regulators of the nucleotide binding, which increase the affinity towards NADPH a 100-fold. Moreover, we showed for the first time that NADH can, like NADPH, form active complexes with Pchlide and POR, however, at much higher concentrations. Additionally, monogalactosyldiacylglycerol (MGDG) was shown to be the main factor responsible for the red shift of the fluorescence emission maximum of Pchlide:POR:NADPH complexes. Importantly, the emission maximum at 654 nm was obtained only for the reaction mixtures supplemented with MGDG and at least one of the negatively charged plant lipids. Moreover, the site-directed mutagenesis allowed us to identify amino acid residues that may be responsible for lipid binding and Pchlide coordination. Our experiments allowed us to identify six different Pchlide:POR complexes that differ in the fluorescence emission maxima of the pigment. The results presented here reveal the contribution of thylakoid lipids in the regulation of the chlorophyll biosynthesis pathway; however, the molecular mechanisms of this process are to be clarified.

scholarly journals Identification of Residues Important for the Activity ofHaloferax volcaniiAglD, a Component of the Archaeal N-Glycosylation Pathway

Archaea ◽  
2010 ◽  
Vol 2010 ◽  
pp. 1-9 ◽  
Author(s):  
Lina Kaminski ◽  
Jerry Eichler
Keyword(s):  
Biochemical Characterization ◽  
Haloferax Volcanii ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Natural Substrate ◽  
Identify Amino Acid ◽  
Glycosylation Pathway

InHaloferax volcanii, AglD adds the final hexose to the N-linked pentasaccharide decorating the S-layer glycoprotein. Not knowing the natural substrate of the glycosyltransferase, together with the challenge of designing assays compatible with hypersalinity, has frustrated efforts at biochemical characterization of AglD activity. To circumvent these obstacles, an in vivo assay designed to identify amino acid residues important for AglD activity is described. In the assay, restoration of AglD function in anHfx. volcanii aglDdeletion strain transformed to express plasmid-encoded versions of AglD, generated through site-directed mutagenesis at positions encoding residues conserved in archaeal homologues of AglD, is reflected in the behavior of a readily detectable reporter of N-glycosylation. As such Asp110 and Asp112 were designated as elements of the DXD motif of AglD, a motif that interacts with metal cations associated with nucleotide-activated sugar donors, while Asp201 was predicted to be the catalytic base of the enzyme.

scholarly journals Mutational Analysis of the Tup1 General Repressor of Yeast

Genetics ◽  
1998 ◽  
Vol 148 (2) ◽  
pp. 637-644
Author(s):  
Pauline M Carrico ◽  
Richard S Zitomer
Keyword(s):  
Mating Type ◽  
Transcriptional Repression ◽  
Mutational Analysis ◽  
Site Directed Mutagenesis ◽  
Missense Mutations ◽  
Amino Acid Residues ◽  
Repeat Region ◽  
Identify Amino Acid ◽  
Repression Complex ◽  
Mating Type Genes

Abstract The Tup1 and Ssn6 proteins of Saccharomyces cerevisiae form a general transcriptional repression complex that regulates the expression of a diverse set of genes including aerobically repressed hypoxic genes, a-mating type genes, glucose repressed genes, and genes controlling cell flocculence. To identify amino acid residues in the Tup1 protein that are required for repression function, we selected for mutations that derepressed the hypoxic genes. Three missense mutations that accumulated stable protein were isolated, and an additional three were generated by site-directed mutagenesis. The mutant protein L62R was unable to complex with Ssn6 or repress expression of reporter genes for the hypoxic and glucose repressed regulons or the flocculence phenotype, however, expression of the a-mating type reporter gene was still repressed. The remaining mutations fell within the WD repeat region of Tup1. These mutations had different effects on the expression of the four Tup1 repressed regulons assayed, indicating that the WD repeats serve different roles for repression of different regulons.

scholarly journals Functional definition of the tobacco protoporphyrinogen IX oxidase substrate-binding site

2007 ◽  
Vol 402 (3) ◽  
pp. 575-580 ◽  
Author(s):  
Ilka U. Heinemann ◽  
Nina Diekmann ◽  
Ava Masoumi ◽  
Michael Koch ◽  
Albrecht Messerschmidt ◽  
...  
Keyword(s):  
Chlorophyll Biosynthesis ◽  
Substrate Binding ◽  
Protoporphyrin Ix ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Enzyme Defects ◽  
Km Value ◽  
Function Relationship ◽  
Definition Of

PPO (protoporphyrinogen IX oxidase) catalyses the flavin-dependent six-electron oxidation of protogen (protoporphyrinogen IX) to form proto (protoporphyrin IX), a crucial step in haem and chlorophyll biosynthesis. The apparent Km value for wild-type tobacco PPO2 (mitochondrial PPO) was 1.17 μM, with a Vmax of 4.27 μM·min−1·mg−1 and a catalytic activity kcat of 6.0 s−1. Amino acid residues that appear important for substrate binding in a crystal structure-based model of the substrate docked in the active site were interrogated by site-directed mutagenesis. PPO2 variant F392H did not reveal detectable enzyme activity indicating an important role of Phe392 in substrate ring A stacking. Mutations of Leu356, Leu372 and Arg98 increased kcat values up to 100-fold, indicating that the native residues are not essential for establishing an orientation of the substrate conductive to catalysis. Increased Km values of these PPO2 variants from 2- to 100-fold suggest that these residues are involved in, but not essential to, substrate binding via rings B and C. Moreover, one prominent structural constellation of human PPO causing the disease variegate porphyria (N67W/S374D) was successfully transferred into the tobacco PPO2 background. Therefore tobacco PPO2 represents a useful model system for the understanding of the structure–function relationship underlying detrimental human enzyme defects.

scholarly journals Spectroscopic and kinetic characterization of the light-dependent enzyme protochlorophyllide oxidoreductase (POR) using monovinyl and divinyl substrates

2006 ◽  
Vol 394 (1) ◽  
pp. 243-248 ◽  
Author(s):  
Derren J. Heyes ◽  
Jerzy Kruk ◽  
C. Neil Hunter
Keyword(s):  
Rhodobacter Capsulatus ◽  
Chlorophyll Biosynthesis ◽  
Kinetic Properties ◽  
Biosynthesis Pathway ◽  
Kinetic Characterization ◽  
Protochlorophyllide Oxidoreductase ◽  
Substrate Analogues ◽  
Steady State Kinetic ◽  
State Kinetic

The enzyme POR [Pchlide (protochlorophyllide) oxidoreductase] catalyses the reduction of Pchlide to chlorophyllide, which is a key step in the chlorophyll biosynthesis pathway. This light-dependent reaction has previously been studied in great detail but recent reports suggest that a mixture of MV (monovinyl) and DV (divinyl) Pchlides may have influenced some of these properties of the reaction. Low-temperature absorbance and fluorescence spectroscopy have revealed several spectral differences between MV and DV Pchlides, which were purified from a Rhodobacter capsulatus strain that was shown to contain a mixture of the two pigments. A thorough steady-state kinetic characterization using both Pchlide forms demonstrates that neither pigment appears to affect the kinetic properties of the enzyme. The reaction has also been monitored following illumination at low temperatures and was shown to consist of an initial photochemical step followed by four ‘dark’ steps for both pigments. However, minor differences were observed in the spectral properties of some of the intermediates, although the temperature dependency of each step was nearly identical for the two pigments. This work provides the first detailed kinetic and spectroscopic study of this unique enzyme using biologically important MV and DV substrate analogues. It also has significant implications for the DV reductase enzyme, which is responsible for converting DV pigments into their MV counterparts, and its position in the sequence of reactions that comprise the chlorophyll biosynthesis pathway.

scholarly journals Site-specific alterations in the B oligomer that affect receptor-binding activities and mitogenicity of pertussis toxin.

1993 ◽  
Vol 177 (1) ◽  
pp. 79-87 ◽  
Author(s):  
Y Lobet ◽  
C Feron ◽  
G Dequesne ◽  
E Simoen ◽  
P Hauser ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Pertussis Toxin ◽  
Neutralizing Antibodies ◽  
Whooping Cough ◽  
Site Directed Mutagenesis ◽  
Molar Ratio ◽  
Amino Acid Residues ◽  
Identify Amino Acid ◽  
Generation Vaccine ◽  
Adp Ribosyltransferase

Pertussis toxin plays a major role in the pathogenesis of whooping cough and is considered an important constituent of vaccines against this disease. It is composed of five different subunits associated in a molar ratio 1S1:1S2:1S3:2S4:1S5. The S1 subunit is responsible for the ADP-ribosyltransferase activity of the toxin. The B moiety, composed of S2 through S5, recognizes and binds to the target cell receptors and has some ADP-ribosyltransferase-independent activities such as mitogenicity. Site-directed mutagenesis of subunits S2 and S3 allowed us to identify amino acid residues involved in receptor binding. Of all the modifications generated, the deletion of Asn 105 in S2 and of Lys 105 in S3 resulted in the more drastic reduction of binding to haptoglobin and CHO cells, respectively. A holotoxin carrying both deletions presented a mitogenicity reduced to an undetectable level. The combination of these B oligomer mutations with two substitutions in the S1 subunit led to the production of a toxin analog with reduced ADP-ribosyltransferase-dependent and -independent activities including mitogenicity. As shown by immunoprecipitation with various monoclonal antibodies, the mutant holotoxin was correctly assembled and antigenically similar to the native toxin. This toxin analog induced toxin-neutralizing antibodies at the same level as the holotoxin carrying only mutations in the S1 subunit, and may therefore be considered a useful candidate for the development of a new generation vaccine against whooping cough.

scholarly journals Identification of residues essential for a two-step reaction by malonyl-CoA synthetase from Rhizobium trifolii

1999 ◽  
Vol 344 (1) ◽  
pp. 159-166 ◽  
Author(s):  
Jae Hyung AN ◽  
Gha Young LEE ◽  
Jin-Won JUNG ◽  
Weontae LEE ◽  
Yu Sam KIM
Keyword(s):  
Enzyme Catalysis ◽  
Hplc Analysis ◽  
Site Directed Mutagenesis ◽  
Soluble Form ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Glutathione S Transferase ◽  
Database Analysis ◽  
Identify Amino Acid ◽  
Malonyl Coa

Malonyl-CoA synthetase (MCS) catalyses the formation of malonyl-CoA in a two-step reaction consisting of the adenylation of malonate with ATP followed by malonyl transfer from malonyl-AMP to CoA. In order to identify amino acid residues essential for each step of the enzyme, catalysis based on chemical modification and database analysis, Arg-168, Lys-170, and His-206 were selected for site-directed mutagenesis. Glutathione-S-transferase-fused enzyme (GST-MCS) was constructed and mutagenized to make R168G, K170M, R168G/K170M and H206L mutants, respectively. The MCS activity of soluble form GST-MCS was the same as that of wild-type MCS. Circular dichroism spectra for the four mutant enzymes were nearly identical to that for the GST-MCS, indicating that Arg-168, Lys-170 and His-206 are not important for conformation but presumably for substrate binding and/or catalysis. HPLC analysis of products revealed that the intermediate malonyl-AMP is not accumulated during MCS catalysis and that none of the mutant enzymes accumulated it either.

A site-directed mutagenesis study to identify amino acid residues involved in the catalytic function of the restriction endonuclease EcoRV

Biochemistry ◽  
1992 ◽  
Vol 31 (20) ◽  
pp. 4808-4815 ◽  
Author(s):  
Ursel Selent ◽  
Thomas Rueter ◽  
Eleonore Koehler ◽  
Michaela Liedtke ◽  
Vera Thielking ◽  
...  
Keyword(s):  
Amino Acid ◽  
Restriction Endonuclease ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Catalytic Function ◽  
Identify Amino Acid ◽  
A Site ◽  
Mutagenesis Study

Molecular Mechanisms of the Inhibitory Effects of Bupivacaine, Levobupivacaine, and Ropivacaine on Sarcolemmal Adenosine Triphosphate–sensitive Potassium Channels in the Cardiovascular System

2004 ◽  
Vol 101 (2) ◽  
pp. 390-398 ◽  
Author(s):  
Takashi Kawano ◽  
Shuzo Oshita ◽  
Akira Takahashi ◽  
Yasuo Tsutsumi ◽  
Yoshinobu Tomiyama ◽  
...  
Keyword(s):  
Cardiovascular System ◽  
Adenosine Triphosphate ◽  
Local Anesthetics ◽  
Molecular Mechanisms ◽  
Transmembrane Domain ◽  
Katp Channels ◽  
Inhibitory Effect ◽  
Amino Acid Residues ◽  
Sulfonylurea Receptor ◽  
Inhibitory Effects

Background Sarcolemmal adenosine triphosphate-sensitive potassium (KATP) channels in the cardiovascular system may be involved in bupivacaine-induced cardiovascular toxicity. The authors investigated the effects of local anesthetics on the activity of reconstituted KATP channels encoded by inwardly rectifying potassium channel (Kir6.0) and sulfonylurea receptor (SUR) subunits. Methods The authors used an inside-out patch clamp configuration to investigate the effects of bupivacaine, levobupivacaine, and ropivacaine on the activity of reconstituted KATP channels expressed in COS-7 cells and containing wild-type, mutant, or chimeric SURs. Results Bupivacaine inhibited the activities of cardiac KATP channels (IC50 = 52 microm) stereoselectively (levobupivacaine, IC50 = 168 microm; ropivacaine, IC50 = 249 microm). Local anesthetics also inhibited the activities of channels formed by the truncated isoform of Kir6.2 (Kir6.2 delta C36) stereoselectively. Mutations in the cytosolic end of the second transmembrane domain of Kir6.2 markedly decreased both the local anesthetics' affinity and stereoselectivity. The local anesthetics blocked cardiac KATP channels with approximately eightfold higher potency than vascular KATP channels; the potency depended on the SUR subtype. The 42 amino acid residues at the C-terminal tail of SUR2A, but not SUR1 or SUR2B, enhanced the inhibitory effect of bupivacaine on the Kir6.0 subunit. Conclusions Inhibitory effects of local anesthetics on KATP channels in the cardiovascular system are (1) stereoselective: bupivacaine was more potent than levobupivacaine and ropivacaine; and (2) tissue specific: local anesthetics blocked cardiac KATP channels more potently than vascular KATP channels, via the intracellular pore mouth of the Kir6.0 subunit and the 42 amino acids at the C-terminal tail of the SUR2A subunit, respectively.

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