358-71-4Relevant articles and documents
IspH-RPS1 and IspH-UbiA: rosetta stone proteins
Rao, Guodong,O'Dowd, Bing,Li, Jikun,Wang, Ke,Oldfield, Eric
, p. 6813 - 6822 (2015/11/24)
The protein IspH, (E)-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate (HMPPP) reductase, is an essential 4Fe-4S cluster-containing protein in the methylerythritol phosphate pathway for isoprenoid biosynthesis. Using a sequence similarity network we found that there are >400 IspH proteins that are about twice as large as most of the IspHs studied to date since their IspH domains are fused to either the ribosomal protein S1 (RPS1), or to a UbiA (4-hydroxybenzoate octaprenyltransferase)-like protein. Many of the IspH-RPS1 proteins are present in anaerobes found in the human gut and some, such as Clostridium botulinum, C. tetani and Fusobacterium nucleatum, are pathogens. The IspH-UbiAs are all found in sulfate-reducing anaerobes. The IspH domains in IspH-RPS1 are fused to 4 and in a few cases 6 tandem repeats in RPS1 that, in most organisms, bind to mRNA or form part of the bacterial ribosome. Mutants in which the four RPS1 domains were sequentially eliminated had similar IspH activity as wild-type protein, indicating they are not essential for IspH catalysis. Overall, the results are of interest since they represent the first isolation of a catalytically active IspH-RPS1, as well as the identification of IspH-UbiA hybrids, two Rosetta stone proteins that are likely to be functionally related - IspH producing the isoprenoids required for a UbiA-like prenyltransferase; the IspH-RPS1 hybrids, perhaps, being involved in the stringent response or as Fe/O2 sensors.
Bioorganometallic mechanism of action, and inhibition, of IspH
Wang, Weixue,Wang, Ke,Liu, Yi-Liang,No, Joo-Hwan,Li, Jikun,Nilges, Mark J.,Oldfield, Eric
body text, p. 4522 - 4527 (2010/10/02)
We have investigated the mechanism of action of Aquifex aeolicus IspH [E-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) reductase], together with its inhibition, using a combination of site-directed mutagenesis (K M;Vmax), EPR and 1H, 2H, 13C, 31P, and 57Fe-electron-nuclear double resonance (ENDOR) spectroscopy. On addition of HMBPP to an (unreactive) E126A IspH mutant, a reaction intermediate forms that has a very similar EPR spectrum to those seen previously with the HMBPP parent molecules, ethylene and allyl alcohol, bound to a nitrogenase FeMo cofactor. The EPR spectrum is broadened on 57Fe labeling and there is no evidence for the formation of allyl radicals. When combined with ENDOR spectroscopy, the results indicate formation of an organometallic species with HMBPP, a π?σ metallacycle or η2-alkenyl complex. The complex is poised to interact with H+ from E126 (and H124) in reduced wt IspH, resulting in loss of water and formation of an η1-allyl complex. After reduction, this forms an η3-allyl π-complex (i.e. containing an allyl anion) that on protonation (at C2 or C4) results in product formation. We find that alkyne diphosphates (such as propargyl diphosphate) are potent IspH inhibitors and likewise form metallacycle complexes, as evidenced by 1H, 2H, and 13C ENDOR, where hyperfine couplings of approximately 6 MHz for 13C and 10 MHz for 1H, are observed. Overall, the results are of broad general interest because they provide new insights into IspH catalysis and inhibition, involving organometallic species, and may be applicable to other Fe4S 4-containing proteins, such as IspG.
Purification and characterization of mouse mevalonate pyrophosphate decarboxylase
Michihara, Akihiro,Akasaki, Kenji,Yamori, Yukio,Tsuji, Hiroshi
, p. 302 - 306 (2007/10/03)
Mevalonate pyrophosphate decarboxylase (MPD) in mouse liver was purified by affinity chromatography. The purified enzyme was a homodimer of 46-kDa subunits and had an isoelectric point of 5.0. Kinetic analysis revealed an apparent Km value of 10 μM for mevalonate pyrophosphate. The enzyme required ATP as a phosphate acceptor and Mg as a divalent cation, which could be substituted with Mn or Co. Its optimum pH was 4.0-7.0. A comparison with MPD from various other sources revealed the mouse MPD to have essentially the same properties as rat MPD, expect for the optimum pH range. An excess of rabbit anti-rat MPD antibody deleted approximately 80% of the MPD activity in the crude extract of mouse liver. These results suggested that the homodimer of 46-kDa subunits represents the major active form of MPD in mice.
