Isoprenoidbiosynthese in pathogenen Bakterien: Nukleare inelastische Streuung ermöglicht Einblicke in den ungewöhnlichen [4Fe‐4S]‐Cluster vom E.‐coli‐Protein LytB/IspH

Abstract

Krankheitsverursachende Mikroorganismen sind zusehends resistent gegen Antibiotikatherapien geworden, während Krankheiten, die als ausgerottet galten, verstärkt wieder auftauchen. Tu berkulose beispielweise ist sogar in den Industrieländern wieder auf dem Vormarsch und verursacht weltweit mehr als 1.1 Millionen Tote pro Jahr.D er Methylerythritolphosphat(MEP)-Weg [1] stellt eine Alternative zum Mevalonatweg [2] dar und wird bei der Biosynthese von Isopentenyldiphosphat (IPP, 1)u nd Dimethylallyldiphosphat (DMAPP, 2)b eschritten. Diese entscheidenden Bausteine sind an der Bildung von essenziellen Te rpenoiden der meisten pathogenen Bakterien (unter anderem Mycobacterium tuberculosis)und Pflanzenplastiden beteiligt. Deswegen gilt der MEP-Weg als Angriffspunkt bei der Entwicklung von neuen antimikrobiellen Stoffen, da er essenziell für Mikroorganismen ist, beim Menschen jedoch nicht vorkommt.[3] Im letzten Schritt dieser Biosyntheseroute wird (E)-4-Hydroxy-3-methylbut-2-en-1-yl-diphosphat (HMBPP, 3) in eine Mischung von IPP (1)u nd DMAPP (2) )a ufweist. [4,5] Dieser Wert ist innerhalb des experimentellen Fehlers identisch mit dem ungewçhnlichen vierten Eisenplatz in der citratgebundenen Form von Aconitase.[6] Ausd iesem Grund wurde vorgeschlagen, dass die Koordinationssphäre dieses speziellen Eisenplatzes drei anorganische Schwefel vom Eisen-Schwefel-Cluster umfasst, sowie zusätzlich drei oder zwei Nicht-Schwefel-Liganden (O und/oder N) in einer Bindungsart, die ähnlich zu der von substratgebundener Aconitase ist. [4,6] Erste Rçntgenstrukturanalysen von substratfreiem LytB von Aquifex aeolicus [7] und Escherichia coli [8]

Further Insight into Crystal Structures of Escherichia coli IspH/LytB in Complex with Two Potent Inhibitors of the MEP Pathway: A Starting Point for Rational Design of New Antimicrobials

“…Therefore, the alternative conformation reported for 12 is most probably a result of the absence of the apical iron that led to the formation of an ammonium–carboxylate ion pair with E126 for a fraction of AMBPP in the active site. The lower occupancy of the apical iron in the published structure (PDB ID: 4H4D) might also explain the discrepancies that were observed in the fitting of the NRVS data …”

“…This oxygen‐sensitive protein harbors a peculiar [4Fe−4S] 2+ center, with one iron linked to three inorganic sulfur atoms from the cluster and to two or three non‐sulfur ligands (N and/or O), as shown by Mössbauer spectroscopic studies of the substrate‐free enzyme . Very recently, synchrotron‐based nuclear resonance vibrational spectroscopy (NRVS) experiments, a technique that specifically senses vibrations involving iron, in combination with quantum chemical–molecular mechanical (QM/MM) calculations, provided evidence that these unknown ligands are three water molecules …”

IspH–RPS1 and IspH–UbiA: “Rosetta stone” proteins

“… 5 IspH is a 4Fe–4S cluster-containing protein 6 that catalyzes the 2H + /2e – reductive dehydroxylation of HMBPP into a ∼1 : 5 mixture of dimethylallyl diphosphate and isopentyl diphosphate. Its structure and mechanism of action have been studied extensively, both experimentally 7 – 13 and computationally, 14 – 16 and several inhibitors have been developed. 7 The mechanism of action is remarkable and the consensus view now is that IspH utilizes a bio-organometallic mechanism in which an allyl species binds to the Fe–S cluster.…”

Methylerythritol Phosphate Pathway: Enzymatic Evidence for a Rotation in the LytB/IspH‐Catalyzed Reaction

“…IspH catalyzes this reductive dehydroxylation by using an oxygen sensitive [4Fe‐4S] 2+ cluster. Mössbauer spectroscopy and, more recently, nuclear resonance vibrational spectroscopy experiments revealed that the [4Fe‐4S] 2+ cluster of substrate‐free IspH is peculiar as one of its four iron sites is in an octahedral coordination geometry, with three inorganic sulfur atoms of the iron sulfur cluster and three water molecules as ligands . This unusual coordination with labile ligands is at the origin of the instability of IspH [4Fe‐4S] cluster.…”

“…This unusual coordination with labile ligands is at the origin of the instability of IspH [4Fe‐4S] cluster. It is now well established that the first step in the IspH mechanism is the binding of the OH group of the substrate to this apical iron accompanied by the release of water molecules . The IspH structure in complex with HMBPP showed that the OH group not only binds the apical iron but is also involved in a series of hydrogen bonds involving T167, E126, a water molecule and a phosphate of the substrate .…”