Discovery of acetylene hydratase activity of the iron–sulphur protein IspH

Abstract

The final step of the methylerythritol phosphate isoprenoid biosynthesis pathway is catalysed by the iron–sulphur enzyme IspH, producing the universal precursors of terpenes: isopentenyl diphosphate and dimethylallyl diphosphate. Here we report an unforeseen reaction discovered during the investigation of the interaction of IspH with acetylene inhibitors by X-ray crystallography, Mößbauer, and nuclear magnetic resonance spectroscopy. In addition to its role as a 2H+/2e− reductase, IspH can hydrate acetylenes to aldehydes and ketones via anti-Markovnikov/Markovnikov addition. The reactions only occur with the oxidised protein and proceed via η1-O-enolate intermediates. One of these is characterized crystallographically and contains a C4 ligand oxygen bound to the unique, fourth iron in the 4Fe-4S cluster: this intermediate subsequently hydrolyzes to produce an aldehyde product. This unexpected side to IspH reactivity is of interest in the context of the mechanism of action of other acetylene hydratases, as well as in the design of antiinfectives targeting IspH.

Spectroscopic and Computational Investigations of Ligand Binding to IspH: Discovery of Non‐diphosphate Inhibitors

“…We also found no spectral shifts when a uniformly 13 C-labeled analog of PPP ( 5 ) was bound to the protein (blue line, Figure 1C). These results are consistent with the presence of a single water molecule [14] binding to the 4 th Fe—the tetrahedral geometry seen in the other IspH structures with 2, 6 and 7 [16] —with no significant bonding between the alkyne group and the cluster. A compilation of the NRVS spectra of all of the variously ligated protein and model compound 4Fe-4S cluster-containing systems discussed above, highlighting their similarities, is shown in Supporting Information Figure S1.…”

“…Plus, the 1-amino ( 6 ) and 1-thio ( 7 ) analogs of HMBPP have been found to inhibit IspH with K i ~20–50 nM [11–12] , and we [13] and others (PDB ID codes: 3ZGL, 3ZGN) have reported their X-ray structures which are basically the same as found with the HMBPP substrate, with N,S binding to the unique, 4 th Fe. We also reported [14] several other IspH-ligand complex structures with, in each case, a single O-containing ligand (alcoholate or enolate) bound to the unique, 4 th Fe atom with Fe-O bond lengths of ~2 Å. What has been (and still is) missing is the X-ray structure of a “ligand free” IspH-either reduced ([Fe 4 S 4 ] + ) or oxidized ([Fe 4 S 4 ] 2+ ), the problem being that it has not been possible to crystallize the 4-Fe containing protein.…”

Advanced paramagnetic resonance spectroscopies of iron–sulfur proteins: Electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM)

“…Through the combined methods of crystallography and advanced EPR, Groll and Oldfield have made additional studies of the inhibitors of IspG and IspH and their organometallic binding modes. [215–219]…”

Biometallorganische Chemie mit IspG und IspH: Struktur, Funktion und Hemmung der an der Isoprenoid‐Biosynthese beteiligten [Fe4S4]‐Proteine

“…[91] Im Fall von 22 enthüllte die Kristallstruktur wieder eine chemische Reaktion der Alkingruppe (Abbildung 11 c, PDB: 3UTD), aber anstelle eines Aldehyds wiesen die Rçntgendaten auf eine Markownikow-Addition und Bildung des Ketons 30. [91] Die von IspH katalysierten Reaktionen sind in Schema 13 zusammengefasst. Alle Substrate enthalten eine Diphosphatgruppe, die an die konservierte Diphosphat-Bindungsstelle bindet, [19,47] [5] Im Fall der Fumarase A (FumA) aus E. coli ist das Enzym auch in der Lage, die Hydratation eines Acetylens, Alcetylendicarboxylat, [5,92,93] [98] Ebenso sind die Enzyme Dihydroxysäure-Dehydratase (DHAD) [99] und Isopropylmalat-Isomerase (IPMI) [100] Ziele, um die Biosynthese verzweigtkettiger Aminosäuren in Tuberkulosebakterien (in Makrophagen) zu hemmen und sind als Angriffsstellen für Herbizide interessant; verschiedene Inhibitoren wurden beschrieben.…”