Surprisingly uninhibited: The inhibition of hydrogenases by oxygen is intensely studied because this is the main obstacle to using these enzymes in biofuel cells. The hydrogenase from Clostridium acetobutylicum (see structure) was found to react surprisingly slowly with O2. The inhibition mechanism was elucidated and the kinetics were quantitatively defined. This is a prerequisite for improving the enzyme further by genetic engineering and for assessing its potential in technological devices.

NifB-co, a Fe-S cluster produced by the enzyme NifB, is an intermediate on the biosynthetic pathway for the iron molybdenum cofactor (FeMo-co) of nitrogenase. We have used Fe K-edge extended xray absorption fine structure (EXAFS) spectroscopy together with 57 Fe nuclear resonance vibrational spectroscopy (NRVS) to probe the structure of NifB-co while bound to the NifX protein from Azotobacter vinelandii. EXAFS analysis of the NifX:NifB-co complex yields Fe-S distances of 2.26 Å and Fe-Fe distances of 2.66 Å and 3.74 Å. Search profile analyses reveals the presence of a single Fe-N (or C, O) interaction at 2.04 Å, implying that the interstitial light atom proposed to be present in FeMo-co is already inserted into NifB-co. NRVS reveals strong bands from Fe-S bending and stretching modes peaking around 270, 315, 385, and 408 cm −1 . Additional intensity at ~185 -200 cm −1 is interpreted as a set of cluster 'breathing' modes similar to those seen for the FeMo-cofactor. The strength and location of these modes also strongly suggest that the FeMo-co interstitial light atom seen in the crystal structure is already in place in NifB-co. Both the EXAFS and NRVS data are best simulated using a Fe 6 S 9 X trigonal prism structure analogous to the 6Fe core of FeMo-co, although a 7Fe structure made by capping one trigonal 3S terminus with Fe cannot be ruled out. This implies that the interstitial light atom is already present at an early stage in FeMo-co biosynthesis prior to the incorporation of Mo and R-homocitrate.

In Clostridium acetobutylicum, [FeFe]-hydrogenase is involved in hydrogen production in vivo by transferring electrons from physiological electron donors, ferredoxin and flavodoxin, to protons. In this report, by modifications of the purification procedure, the specific activity of the enzyme has been improved and its complete catalytic profile in hydrogen evolution, hydrogen uptake, proton/deuterium exchange and para-H2/ortho-H2 conversion has been determined. The major ferredoxin expressed in the solvent-producing C. acetobutylicum cells was purified and identified as encoded by ORF CAC0303. Clostridium acetobutylicum recombinant holoflavodoxin CAC0587 was also purified. The kinetic parameters of C. acetobutylicum [FeFe]-hydrogenase for both physiological partners, ferredoxin CAC0303 and flavodoxin CAC0587, are reported for hydrogen uptake and hydrogen evolution activities.

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When employing biotechnological processes for the procurement of biofuels and bio-products from microalgae, one of the most critical steps affecting economy and yields is the "cell disruption" stage. Currently, enzymatic cell disruption has delivered effective and cost competitive results when compared to mechanical and chemical cell disruption methods. However, the introduction of enzymes implies additional associated cost within the overall process. In order to reduce this cost, autolysis of microalgae is proposed as alternative enzymatic cell disruption method. This review aims to provide the state of the art of enzymatic cell disruption treatments employed in biorefinery processes and highlights the use of endopeptidases. During the enzymatic processes of microalgae life cycle, some lytic enzymes involved in cell division and programmed cell death have been proven useful in performing cell lysis. In this context, the role of endopeptidases is emphasized. Mirroring these natural events, an alternative cell disruption approach is proposed and described with the potential to induce the autolysis process using intrinsic cell enzymes. Integrating induced autolysis within biofuel production processes offers a promising approach to reduce overall global costs and energetic input associated with those of current cell disruption methods. A number of options for further inquiry are also discussed.

The establishment of polarity is a critical process in pathogenic fungi, mediating infection-related morphogenesis and host tissue invasion. Here, we report the identification of TPC1 (Transcription factor for Polarity Control 1), which regulates invasive polarized growth in the rice blast fungus Magnaporthe oryzae. TPC1 encodes a putative transcription factor of the fungal Zn(II)2Cys6 family, exclusive to filamentous fungi. Tpc1-deficient mutants show severe defects in conidiogenesis, infection-associated autophagy, glycogen and lipid metabolism, and plant tissue colonisation. By tracking actin-binding proteins, septin-5 and autophagosome components, we show that Tpc1 regulates cytoskeletal dynamics and infection-associated autophagy during appressorium-mediated plant penetration. We found that Tpc1 interacts with Mst12 and modulates its DNA-binding activity, while Tpc1 nuclear localisation also depends on the MAP kinase Pmk1, consistent with the involvement of Tpc1 in this signalling pathway, which is critical for appressorium development. Importantly, Tpc1 directly regulates NOXD expression, the p22phox subunit of the fungal NADPH oxidase complex via an interaction with Mst12. Tpc1 therefore controls spatial and temporal regulation of cortical F-actin through regulation of the NADPH oxidase complex during appressorium re-polarisation. Consequently, Tpc1 is a core developmental regulator in filamentous fungi, linking the regulated synthesis of reactive oxygen species and the Pmk1 pathway, with polarity control during host invasion.

The biological activation of N2 occurs at the FeMo-cofactor, a 7Fe-9S-Mo-C-homocitrate cluster. FeMo-cofactor formation involves assembly of a Fe6-8 -SX -C core precursor, NifB-co, which occurs on the NifB protein. Characterization of NifB-co in NifB is complicated by the dynamic nature of the assembly process and the presence of a permanent [4Fe-4S] cluster associated with the radical SAM chemistry for generating the central carbide. We have used the physiological carrier protein, NifX, which has been proposed to bind NifB-co and deliver it to the NifEN protein, upon which FeMo-cofactor assembly is ultimately completed. Preparation of NifX in a fully NifB-co-loaded form provided an opportunity for Mössbauer analysis of NifB-co. The results indicate that NifB-co is a diamagnetic (S=0) 8-Fe cluster, containing two spectroscopically distinct Fe sites that appear in a 3:1 ratio. DFT analysis of the (57) Fe electric hyperfine interactions deduced from the Mössbauer analysis suggests that NifB-co is either a 4Fe(2+) -4Fe(3+) or 6Fe(2+) -2Fe(3+) cluster having valence-delocalized states.

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