Structure of the Mg-Chelatase Cofactor GUN4 Reveals a Novel Hand-Shaped Fold for Porphyrin Binding

In plants, the accumulation of the chlorophyll precursor Mg-protoporphyrin IX (Mg-Proto) in the plastid regulates the expression of a number of nuclear genes with functions related to photosynthesis. Analysis of the plastid-to-nucleus signaling activity of Mg-Proto in Arabidopsis thaliana led to the discovery of GUN4, a novel porphyrin-binding protein that also dramatically enhances the activity of Mg-chelatase, the enzyme that synthesizes Mg-Proto. GUN4 may also play a role in both photoprotection and the cellular shuttling of tetrapyrroles. Here we report a 1.78-Å resolution crystal structure of Synechocystis GUN4, in which the porphyrin-binding domain adopts a unique three dimensional fold with a “cupped hand” shape. Biophysical and biochemical analyses revealed the specific site of interaction between GUN4 and Mg-Proto and the energetic determinants for the GUN4 • Mg-Proto interaction. Our data support a novel protective function for GUN4 in tetrapyrrole trafficking. The combined structural and energetic analyses presented herein form the physical-chemical basis for understanding GUN4 biological activity, including its role in the stimulation of Mg-chelatase activity, as well as in Mg-Proto retrograde signaling

The Heme Biosynthetic Pathway of the Obligate Wolbachia Endosymbiont of Brugia malayi as a Potential Anti-filarial Drug Target

Human filarial nematodes are causative agents of elephantiasis and African river blindness, which are among the most debilitating tropical diseases. Currently used drugs mainly affect microfilariae (mf) and have less effect on adult filarial nematodes, which can live in the human host for more than a decade. Filariasis drug control strategy relies on recurrent mass drug administration for many years. Development of novel drugs is also urgently needed due to the threat of drug resistance occurrence. Most filarial worms harbor an obligate endosymbiotic bacterium, Wolbachia, whose presence has been identified as a potential drug target. Comparative genomics had suggested Wolbachia heme biosynthesis as a potential drug target, and we present an analysis of selected enzymes alongside their human homologues from several different aspects—gene phylogenetic analyses, in vitro enzyme kinetic and inhibition assays and heme-deficient E. coli complementation assays. We also conducted ex vivo Brugia malayi viability assays using heme pathway inhibitors. These experiments demonstrate that heme biosynthesis could be critical for filarial worm survival and thus is a potential anti-filarial drug target set

Opportunities for Energy Efficiency Improvement in a Renewable Fuels Process

A larger part of the cooling and heating demand in a renewable fuels plant is accomplished by using air-cooling and steam in several alternating steps. This study examine what opportunities there are for energy efficiency improvements on site, in order to reduce the electricity and fuel consumption in the plant. The evaluation was done by using pinch tools on three different case studies: theoretical case based on technical specifications, operational case based on averaged measurements from the process and the adjusted operational case with changed temperature targets respectively. The investigated cases had total heating demands of 18.7 MW, 20.0 MW and 19.2 MW and total cooling demands of 20.4 MW, 18.9 MW and 17.7 MW respectively. Results from the pinch analysis of the theoretical case indicated that there were three ways to improve energy efficiency in the plant: by removal of pinch rule violations, by optimizing the temperature level at which utility was supplied or increasing methanol condensation temperatures in an integrated HEN (i.e. raising saturation pressure). Actual temperatures and flows in the operational case were analysed. By changing target temperatures the plant could save 2 123 kW of energy, both from heating and electricity. Assuming the operation would be adjusted accordingly, the adjusted operational case was created. On this case the full pinch analysis with retrofit suggestions was performed, as well as investigating the full potential of optimizing hot utility levels and condensing methanol at a higher temperature. By performing a retrofit of the existing HEN, 1 517 kW can be heat integrated. This would save the plant 5.54 MSEK annually in reduced utility costs. By heating the two flows entering the reactors with MP steam before using HP steam, 5 100 kW could be saved. This shift of hot utility level would save the plant 3.74 MSEK annually in reduced steam costs. By building two new flashing steps at 8 bar, which would supply condensing methanol at 135ºC, approximately 2 194 kW would become available. If integrated in such a way that it could replace the corresponding load in MP steam, 15.5 MSEK in eliminated steam costs would be saved annually. It was recommended that options for flashing methanol at intermediate pressures, combined with a new retrofit investigation, would be the best alternative to investigated further

Crystallographic studies on porphyrin metallation by ferrochelatase.

Ferrochelatase catalyses the terminal step in heme biosynthesis by inserting a ferrous ion into protoporphyrin IX. The reaction mechanism has in this thesis been studied by mainly crystallographic methods. The structure of ferrochelatase from Bacillus subtilis co-crystallized with N-methyl mesoporphyrin allowed for the first time a characterization of the active site of the enzyme. The structure suggests a mechanism of porphyrin distortion where the pyrrole rings B, C and D are fixed by the enzyme, while pyrrole ring A is forced to tilt 36?. Soakings of Zn2+ into ferrochelatase crystals showed that the metal was coordinated to the invariant residues His183 and Glu264 located in the active site. These residues are proposed to participate in a sitting-atop complex with the metal and porphyrin. A fully hydrated Mg2+ complex was found located 7 Å away from the Zn2+ ion. Mg2+ had a stimulatory effect on the enzyme activity. From biochemical and theoretical studies it was found that the two metals interact with each other, resulting in lower binding affinity for Zn2+. The structure of ferrochelatase from Saccharomyces cerevisiae was determined to a resolution of 2.4 Å. It was found to be a homodimer. Metal soakings with a substrate metal (Co2+) and an inhibitor (Cd2+) showed that they were bound to the same site as was observed earlier for ferrochelatase from B. subtilis. The observation that the two monomers bind metal differently suggest that porphyrin metallation in the yeast monomers occur in an alternating fashion, like a two-stroke engine

Crystallographic studies on porphyrin metallation by ferrochelatase

Popular Abstract in Swedish Proteiner är livsnödvändiga och finns i alla celler. De flesta proteinerna katalysear kemiska reaktioner och kallas då enzymer. Enzymer och proteiner (äggviteämnen) är uppbyggda av aminosyror ihopkopplade till kedjor som kan vara olika långa. Det finns 20 stycken olika aminosyror. Man kan göra en liknelse med ett pärlhalsband tillverkat av 20 olika pärlor, varje pärla med var sin färg. Då blir proteinkedjan ett långt pärlhalsband med färger som kommer efter varandra i en till synes slumpvis ordning. Men ordningen samt längden på kedjan är inte slumpvis i cellerna, den styrs av generna. Generna kan sägas vara cellens arkiv, där enstaka kopior kan hämtas ut på ritningar för hur proteinerna ska vara sammansatta. Kopiorna används av cellens speciella maskiner för proteinproduktion, ribosomerna. Ordningen av aminosyrorna och längden på kedjan bestämmer hur protein kedjan veckar sig och hur strukturen således kommer att se ut. Om strukturen är känd kan man få viktiga ledtrådar för att kunna designa läkemedel rätt. Man kan visserligen ta reda på vad ett visst enzym gör och när det gör det, men inte hur enzymet katalyserar en reaktion, om inte dess struktur är känd. Röntgenkristallografi och proteinkristaller Strukturbestämning kan göras antingen med NMR-teknik (Nuclear magnetic resonance) eller med röntgenkristallografi, som jag har använt mig av. Metoden bygger på att man låter växa kristaller av proteinet man vill strukturbestämma. En kristall är sammansatt av miljarder av molekyler som sitter i ett regelbundet tredimensionellt mönster, på samma vis som natrium och kloridjonerna bygger upp vanligt koksalt. Proteinkristaller kan man få att bildas när man låter sin koncentrerade proteinlösning under vissa kemiska betingelser långsamt torka, fastän inte torka ut helt. Det kan ibland ta tid att hitta de speciella kemiska betingelserna, eftersom olika protein kräver olika betingelser för att kristallisera. Med lite tur och skicklighet kan man få fram kristaller stora nog för att man ska kunna undersöka dem och exponera dem för en stark och koncentrerad röntgenstråle. I kristallografin utnyttjar man ett fenomen som kallas diffraktion. Fenomenet medför att röntgenstrålar som passerar en proteinkristall sprids åt olika håll, som om det fanns osynliga små spegelplan som reflekterade röntgenstrålarna i kristallen. Mönstret av de spridda röntgenstrålarna som uppkommer efter att ha gått igenom kristallen tas tillvara. De erhållna mätdata databehandlas med hjälp av matematiska modeller som beskriver hur kristaller geometriskt är uppbyggda. Eftersom det är elektronerna från alla atomer i kristallen som tillsammans sprider röntgenstrålarna är det i slutändan ett tredimensionellt elektronmoln man får ut efter strukturbestämningen. Med ett tydligt tredimensionellt elektronmoln kan man passa in proteinet bit för bit, aminosyra för aminosyra. På så vis får man fram, förenklat beskrivet, en modell för proteinstrukturen. Ferrochelatase tillverkar heme Jag har studerat enzymet som katalyserar reaktionen då en järn-atom sätts på plats i en liten molekyl som kallas protoporfyrin så att heme bildas. Det är samma heme som behövs för kroppens produktion av hemoglobin. Enzymet som utför denna reaktion heter ferrochelatase. En defekt i genen för ferrochelatas leder till en blodsjukdom med symptom som ljuskänslig hy och leverskador. Hemoglobin finns i de röda blodkropparna i blodet och har till uppgift att transportera syre till alla kroppens celler. En vuxen man har 200 miljarder röda blodkroppar. De har en livslängd på ca 120 dagar, och varje minut behövs därför över 1 miljon nya röda blodkroppar produceras. Denna enorma produktion sker med hjälp av enzymet ferrochelatas. Reaktionsmekanismen börjar klarna Med ledning av strukturella data av enzymet tillsammans med olika metaller (tex koppar, cobolt, cadmium och zink) och en hemeliknande molekyl, har jag studerat hur och var de båda substraten binder in till proteinet. På så sätt har man kunnat skapa en bild av de olika stegen i reaktionsmekanismen.Det visade sig att enzymet fungerar som en magnet och "drar in" järn-atomen till det aktiva sätet i enzymet där reaktionen äger rum. Den blivande hememolekylen böjs dessutom så att järnatomen lättare kan "hoppa in". När järnet sitter på plats lossnar heme för att det inte längre passar så bra in i enzymets bindningsficka. Då blir det plats för en ny reaktioncykel och så håller det på. Hemet transporteras vidare av kroppens transportmaskineri och sätts ihop med ett protein så att hemoglobin bildas till nytta för syretransporten. Dessa rön om hur en metall sätts in med hjälp av enzym kan ge viktiga ledtrådar för hur andra liknande molekyler som innehåller metall bildas, tex klorofyll i växter, (då magnesium sätts in) och vitamin B12 (då cobolt sätts in).Ferrochelatase catalyses the terminal step in heme biosynthesis by inserting a ferrous ion into protoporphyrin IX. The reaction mechanism has in this thesis been studied by mainly crystallographic methods. The structure of ferrochelatase from Bacillus subtilis co-crystallized with N-methyl mesoporphyrin allowed for the first time a characterization of the active site of the enzyme. The structure suggests a mechanism of porphyrin distortion where the pyrrole rings B, C and D are fixed by the enzyme, while pyrrole ring A is forced to tilt 36?. Soakings of Zn2+ into ferrochelatase crystals showed that the metal was coordinated to the invariant residues His183 and Glu264 located in the active site. These residues are proposed to participate in a sitting-atop complex with the metal and porphyrin. A fully hydrated Mg2+ complex was found located 7 Å away from the Zn2+ ion. Mg2+ had a stimulatory effect on the enzyme activity. From biochemical and theoretical studies it was found that the two metals interact with each other, resulting in lower binding affinity for Zn2+. The structure of ferrochelatase from Saccharomyces cerevisiae was determined to a resolution of 2.4 Å. It was found to be a homodimer. Metal soakings with a substrate metal (Co2+) and an inhibitor (Cd2+) showed that they were bound to the same site as was observed earlier for ferrochelatase from B. subtilis. The observation that the two monomers bind metal differently suggest that porphyrin metallation in the yeast monomers occur in an alternating fashion, like a two-stroke engine