Mark Emptage

Purification and characterization of a 7Fe ferredoxin from Streptomyces griseus

A ferredoxin has been purified from Streptomyces griseus grown in soybean flour-containing medium. The homogeneous protein has a molecular weight near 14,000 as determined by both PAGE and size exclusion chromatography. The iron and labile sulfide content is 6-7 atoms/mole protein. EPR spectroscopy of native S. griseus ferredoxin shows an isotropic signal at g = 2.01 which is typical of [3Fe-4S]1+ clusters and which quantitates to 0.9 spin/mole. Reduction of the ferredoxin by excess dithionite at pH 8.0 produces an EPR silent state with a small amount of a g = 1.95 type signal. Photoreduction in the presence of deazaflavin generates a signal typical of [4Fe-4S]1+ clusters at much higher yields (0.4-0.5 spin/mole) with major features at g-values of 2.06, 1.94, 1.90 and 1.88. This latter EPR signal is most similar to that seen for reduced 7Fe ferredoxins, which contain both a [3Fe-4S] and [4Fe-4S] cluster. In vitro reconstitution experiments demonstrate the ability of the S. griseus ferredoxin to couple electron transfer between spinach ferredoxin reductase and S. griseus cytochrome P-450soy for NADPH-dependent substrate oxidation. This represents a possible physiological function for the S. griseus ferredoxin, which if true, would be the first functional role demonstrated for a 7Fe ferredoxin.

Primary structure of a 7Fe ferredoxin from Streptomyces griseus

The complete primary structure of a Streptomyces griseus (ATCC 13273) 7Fe ferredoxin, which can couple electron transfer between spinach ferredoxin reductase and S. griseus cytochrome P-450soy for NADPH-dependent substrate oxidation, has been determined by Edman degradation of the whole protein and peptides derived by Staphylococcus aureus V8 proteinase and trypsin digestion. The protein consists of 105 amino acids and has a calculated molecular weight, including seven irons and eight sulfurs, of 12,291. The ferredoxin sequence is highly homologous (73%) to that of the 7Fe ferredoxin from Mycobacterium smegmatis. The N-terminal half of the sequence, which is the Fe-S clusters binding domain, has more than 50% homology with other 7Fe ferredoxins. In particular, the seven cysteines known from the crystal structure of Azotobacter vinelandii ferredoxin I to be involved in binding the two Fe-S clusters are conserved.

Cloning, expression, purification and crystallization of saccharopine reductase from Magnaporthe grisea.

The gene coding for saccharopine reductase (E.C. 1.5.1.10), an enzyme of the alpha-aminoadipic pathway of lysine biosynthesis in the pathogenic fungus Magnaporthe grisea, was cloned and expressed in Escherichia coli. The purified enzyme was crystallized in space groups C2 and C222(1) using ammonium sulfate pH 4.8 or PEG 6000 pH 4. 1 as precipitants. The unit-cell parameters are a = 115.0, b = 56.6, c = 74.3 A, beta = 111.1 degrees for space group C2, and a = 89.3, b = 119.0, c = 195.9 A for space group C222(1). The crystals diffract to resolutions of 2.0 A (C2) and 2.4 A (C222(1)) at synchrotron sources.

Sessions 2 and 7: advances in microbial science and technology.

The Microbial Science and Technology Sessions emphasized a variety of approaches used for development of superior recombinant microorganisms for fermentation of multiple sugars typically present in any lignocellulosic hydrolyzate to fuels and chemicals. Efficient conversion of lignocellulosic biomass to liquid transportation fuels is a major goal for many in these sessions. The Session 2 contained six oral presentations and 62 poster presentations and Session 7 contained an additional six oral presentations and 49 posters. A brief overview of each oral Session and a summary of topics presented during the poster sessions are provided below. Session 2 opened with a comparison of different pretreatments for corn stover, followed by fermentation using different recombinant organisms. Next, the metabolic engineering of a novel thermophilic Geobacillus sp. capable of high-yield ethanol production was discussed. The pathway for D-galacturonic acid catabolism in fungi was elucidated in the third presentation and determined to be different from any previously described pathway. Butanol production from engineered Escherichia coli and improvements in solventogenic Clostridium butanol production were the topics for another presentation. Metabolic pathway engineering of Thermoanaerobacterium saccharolyticum for efficient ethanol production and reduction of cellulase loading emphasized a consolidated bioprocessing approach. New methods for isolating novel biocatalysts and enzymes from lignocellulosedegrading insects rounded out this first session. Appl Biochem Biotechnol (2009) 153:11–12 DOI 10.1007/s12010-009-8609-9