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Assembly of Multiple CotC Forms into the Bacillus subtilis Spore Coat. Rachele Isticato, 2004.We report evidence that the CotC polypeptide, a previously identified component of the Bacillus subtilis spore coat, is assembled into at least four distinct forms . Two of these, having molecular masses of 12 and 21 kDa, appeared 8 h after the onset of sporulation and were probably assembled on the forming spore immediatelyafter their synthesis, since no accumulation of either of themwas detected in the mother cell compartment, where their synthesisoccurs . The other two components, 12.5 and 30 kDa, were generated2 h later and were probably the products of posttranslationalmodifications of the two early forms occurring directly on thecoat surface during spore maturation . None of the CotC formswas found either on the spore coat or in the mother cell compartmentof a cotH mutant . This indicates that CotH serves a dual roleof stabilizing the early forms of CotC and promoting the assemblyof both early and late forms on the spore surface. The 1.3-Angstrom-Resolution Crystal Structure of ß-Ketoacyl-Acyl Carrier Protein Synthase II from Streptococcus pneumoniae. Allen C. Price, 2003.The ß-ketoacyl-acyl carrier protein synthases are members of the thiolase superfamily and are key regulators of bacterial fatty acid synthesis . As essential components of the bacterial lipid metabolic pathway, they are an attractive target for antibacterial drug discovery . We have determined the 1.3 Å resolution crystal structure of the ß-ketoacyl-acyl carrier protein synthase II (FabF) from the pathogenic organism Streptococcus pneumoniae . The protein adopts a duplicated ß  ßßßß fold, which is characteristic of the thiolase superfamily . The two-fold pseudosymmetry is broken by the presence of distinct insertions in the two halves of the protein . These insertions have evolved to bind the specific substrates of this particular member of the thiolase superfamily . Docking of the pantetheine moiety of the substrate identifies the loop regions involved in substrate binding and indicates roles for specific, conserved residues in the substrate binding tunnel . The active site triad of this superfamily is present in spFabF as His 303, His 337, and Cys 164 . Near the active site is an ion pair, Glu 346 and Lys 332, that is conserved in the condensing enzymes but is unusual in our structure in being stabilized by an Mg2+ ion which interacts with Glu 346 . The active site histidines interact asymmetrically with Lys 332, whose positive charge is closer to His 303, and we propose a specific role for the lysine in polarizing the imidazole ring of this histidine . This asymmetry suggests that the two histidines have unequal roles in catalysis and provides new insights into the catalytic mechanisms of these enzymes .
Plasmid-Encoded Diacetyl (Acetoin) Reductase in Leuconostoc pseudomesenteroides. Fergal P. Rattray, 2003.A plasmid-borne diacetyl (acetoin) reductase (butA) from Leuconostoc pseudomesenteroides CHCC2114 was sequenced and cloned . Nucleotide sequence analysis revealed an open reading frame encoding a protein of 257 amino acids which had high identity at the amino acid level to diacetyl (acetoin) reductases reported previously . Downstream of the butA gene of L . pseudomesenteroides, but coding in the opposite orientation, a putative DNA recombinase was identified . A two-step PCR approach was used to construct FPR02, a butA mutant of the wild-type strain, CHCC2114 . FPR02 had significantly reduced diacetyl (acetoin) reductase activity with NADH as coenzyme, but not with NADPH as coenzyme, suggesting the presence of another diacetyl (acetoin)-reducing activity in L . pseudomesenteroides . Plasmid-curing experiments demonstrated that the butA gene is carried on a 20-kb plasmid in L . pseudomesenteroides . Engineering Redox Cofactor Regeneration for Improved Pentose Fermentation in Saccharomyces cerevisiae. Ritva Verho, 2003.Pentose fermentation to ethanol with recombinant Saccharomyces cerevisiae is slow and has a low yield . A likely reason for this is that the catabolism of the pentoses D-xylose and L-arabinose through the corresponding fungal pathways creates an imbalance of redox cofactors . The process, although redox neutral, requires NADPH and NAD+, which have to be regenerated in separate processes . NADPH is normally generated through the oxidative part of the pentose phosphate pathway by the action of glucose-6-phosphate dehydrogenase (ZWF1) . To facilitate NADPH regeneration, we expressed the recently discovered gene GDP1, which codes for a fungal NADP+-dependent D-glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPDH) (EC 1.2.1.13), in an S . cerevisiae strain with the D-xylose pathway . NADPH regeneration through an NADP-GAPDH is not linked to CO2 production . The resulting strain fermented D-xylose to ethanol with a higher rate and yield than the corresponding strain without GDP1; i.e., the levels of the unwanted side products xylitol and CO2 were lowered . The oxidative part of the pentose phosphate pathway is the main natural path for NADPH regeneration . However, use of this pathway causes wasteful CO2 production and creates a redox imbalance on the path of anaerobic pentose fermentation to ethanol because it does not regenerate NAD+ . The deletion of the gene ZWF1 (which codes for glucose-6-phosphate dehydrogenase), in combination with overexpression of GDP1 further stimulated D-xylose fermentation with respect to rate and yield . Through genetic engineering of the redox reactions, the yeast strain was converted from a strain that produced mainly xylitol and CO2 from D-xylose to a strain that produced mainly ethanol under anaerobic conditions .
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