Mass Determination of Entire Amyloid Fibrils by Using Mass Spectrometry.

Amyloid fibrils are self-assembled protein structures with important roles in biology (either pathogenic or physiological), and are attracting increasing interest in nanotechnology. However, because of their high aspect ratio and the presence of some polymorphism, that is, the possibility to adopt various structures, their characterization is challenging and basic information such as their mass is unknown. Here we show that charge-detection mass spectrometry, recently developed for large self-assembled systems such as viruses, provides such information in a straightforward manner.

The role of the maturase HydG in [FeFe]-hydrogenase active site synthesis and assembly.

[FeFe]-hydrogenases catalyze the protons/hydrogen interconversion through a unique di-iron active site consisting of three CO and two CN ligands, and a non-protein SCH(2)XCH(2)S (X=N or O) dithiolate bridge. Site assembly requires two "Radical-S-adenosylmethionine (SAM or AdoMet)" iron-sulfur enzymes, HydE and HydG, and one GTPase, HydF. The sequence homology between HydG and ThiH, a Radical-SAM enzyme which cleaves tyrosine into p-cresol and dehydroglycine, and the finding of a similar cleavage reaction catalyzed by HydG suggests a mechanism for hydrogenase maturation. Here we propose that HydG is specifically involved in the synthesis of the dithiolate ligand, with two tyrosine-derived dehydroglycines as precursors along with an [FeS] cluster of HydG functioning both as electron shuttle and source of the sulfur atoms.

Mechanism and regulation of the Two-component FMN-dependent monooxygenase ActVA-ActVB from Streptomyces coelicolor.

The ActVA-ActVB system from Streptomyces coelicolor is a two-component flavin-dependent monooxygenase involved in the antibiotic actinorhodin biosynthesis. ActVB is a NADH:flavin oxidoreductase that provides a reduced FMN to ActVA, the monooxygenase that catalyzes the hydroxylation of dihydrokalafungin, the precursor of actinorhodin. In this work, using stopped-flow spectrophotometry, we investigated the mechanism of hydroxylation of dihydrokalafungin catalyzed by ActVA and that of the reduced FMN transfer from ActVB to ActVA. Our results show that the hydroxylation mechanism proceeds with the participation of two different reaction intermediates in ActVA active site. First, a C(4a)-FMN-hydroperoxide species is formed after binding of reduced FMN to the monooxygenase and reaction with O(2). This intermediate hydroxylates the substrate and is transformed to a second reaction intermediate, a C(4a)-FMN-hydroxy species. In addition, we demonstrate that reduced FMN can be transferred efficiently from the reductase to the monooxygenase without involving any protein.protein complexes. The rate of transfer of reduced FMN from ActVB to ActVA was found to be controlled by the release of NAD(+) from ActVB and was strongly affected by NAD(+) concentration, with an IC(50) of 40 microm. This control of reduced FMN transfer by NAD(+) was associated with the formation of a strong charge.transfer complex between NAD(+) and reduced FMN in the active site of ActVB. These results suggest that, in Streptomyces coelicolor, the reductase component ActVB can act as a regulatory component of the monooxygenase activity by controlling the transfer of reduced FMN to the monooxygenase.

X-ray structure of the [FeFe]-hydrogenase maturase HydE from Thermotoga maritima.

Maturation of the [FeFe]-hydrogenase active site depends on at least the expression of three gene products called HydE, HydF, and HydG. We have solved the high resolution structure of recombinant, reconstituted S-adenosine-L-methionine-dependent HydE from Thermotoga maritima. Besides the conserved [Fe(4)S(4)] cluster involved in the radical-based reaction, this HydE was reported to have a second [Fe(4)S(4)] cluster coordinated by three Cys residues. However, in our crystals, depending on the reconstitution and soaking conditions, this second cluster is either a [Fe(2)S(2)] center, with water occupying the fourth ligand site or is absent. We have carried out site-directed mutagenesis studies on the related HydE from Clostridium acetobutylicum, along with in silico docking and crystal soaking experiments, to define the active site region and three anion-binding sites inside a large, positive cavity, one of which binds SCN(-) with high affinity. Although the overall triose-phosphate isomerase-barrel structure of HydE is very similar to that of biotin synthase, the residues that line the internal cavity are significantly different in the two enzymes.

tRNA-modifying MiaE protein from Salmonella typhimurium is a nonheme diiron monooxygenase.

MiaE catalyzes the posttranscriptional allylic hydroxylation of 2-methylthio-N-6-isopentenyl adenosine in tRNAs. The Salmonella typhimurium enzyme was heterologously expressed in Escherichia coli. The purified enzyme is a monomer with two iron atoms and displays activity in in vitro assays. The type and properties of the iron center were investigated by using a combination of UV-visible absorption, EPR, HYSCORE, and Mössbauer spectroscopies which demonstrated that the MiaE enzyme contains a nonheme dinuclear iron cluster, similar to that found in the hydroxylase component of methane monooxygenase. This is the first example of an enzyme from this important class of diiron monooxygenases to be involved in the hydroxylation of a biological macromolecule and the second example of a redox metalloenzyme participating in tRNA modification.