9 research outputs found
Structure and functional characterization of pyruvate decarboxylase from Gluconacetobacter diazotrophicus
BACKGROUND: Bacterial pyruvate decarboxylases (PDC) are rare. Their role in ethanol production and in bacterially
mediated ethanologenic processes has, however, ensured a continued and growing interest. PDCs from Zymomonas
mobilis (ZmPDC), Zymobacter palmae (ZpPDC) and Sarcina ventriculi (SvPDC) have been characterized and ZmPDC
has been produced successfully in a range of heterologous hosts. PDCs from the Acetobacteraceae and their role in
metabolism have not been characterized to the same extent. Examples include Gluconobacter oxydans (GoPDC),
G. diazotrophicus (GdPDC) and Acetobacter pasteutrianus (ApPDC). All of these organisms are of commercial importance.
RESULTS: This study reports the kinetic characterization and the crystal structure of a PDC from Gluconacetobacter
diazotrophicus (GdPDC). Enzyme kinetic analysis indicates a high affinity for pyruvate (KM 0.06 mM at pH 5), high
catalytic efficiencies, pHopt of 5.5 and Topt at 45 degrees C. The enzyme is not thermostable (T of
18 minutes at 60 degrees C) and the calculated number of bonds between monomers and dimers do not give clear indications
for the relatively lower thermostability compared to other PDCs. The structure is highly similar to those described for Z.
mobilis (ZmPDC) and A. pasteurianus PDC (ApPDC) with a rmsd value of 0.57 A for C? when comparing GdPDC to that
of ApPDC. Indole-3-pyruvate does not serve as a substrate for the enzyme. Structural differences occur in two loci,
involving the regions Thr341 to Thr352 and Asn499 to Asp503.
CONCLUSIONS: This is the first study of the PDC from G. diazotrophicus (PAL5) and lays the groundwork for future
research into its role in this endosymbiont. The crystal structure of GdPDC indicates the enzyme to be evolutionarily
closely related to homologues from Z. mobilis and A. pasteurianus and suggests strong selective pressure to keep the
enzyme characteristics in a narrow range. The pH optimum together with reduced thermostability likely reflect the
host organisms niche and conditions under which these properties have been naturally selected for. The lack of activity
on indole-3-pyruvate excludes this decarboxylase as the enzyme responsible for indole acetic acid production in
G. diazotrophicus.IS
The initial and subsequent inflammatory events during calcium oxalate lithiasis
Background: Crystallization is believed to be the initiation step of urolithiasis, even though it is unknown where inside the nephron the first crystal nucleation occurs. Methods: Direct nucleation of calcium oxalate and subsequent events including crystal retention, cellular damage, endocytosis, and hyaluronan (HA) expression, were tested in a two-compartment culture system with intact human proximal tubular HK-2 cell monolayer. Results: Calcium oxalate dihydrate (COD) was nucleated and bound onto the apical surface of the HK-2 cells under hypercalciuric and hyperoxaluric conditions. These cells displayed mild cellular damage and internalized some of the adhered crystals within 18. h post-COD-exposure, as revealed by electron microscopy. Prolonged incubation in complete medium caused significant damage to disrupt the monolayer integrity. Furthermore, hyaluronan disaccharides were detected in the harvested media, and were associated with HAS-3 mRNA expression. Conclusion: Human proximal cells were able to internalize COD crystals which nucleated directly onto the apical surface, subsequently triggering cellular damage and HAS-3 specific hyaluronan synthesis as an inflammatory response. The proximal tubule cells here demonstrate that it plays an important role in facilitating urolithiasis via endocytosis and creating an inflammatory environment whereby free hyaluronan in tubular fluid can act as crystal-binding molecule at the later segments of distal and collecting tubules. © 2010 Elsevier B.V.link_to_subscribed_fulltex
Solution and Gas-Phase H/D Exchange of Protein–Small-Molecule Complexes: Cex and Its Inhibitors
Effective strategy to assign 1H-15N heteronuclear correlation NMR signals from lysine side-chain NH3 + groups of proteins at low temperature
Cloning of a rumen fungal xylanase gene and purification of the recombinant enzyme via artificial oil bodies
The Phylum dictyoglomi
The phylum Dictyoglomi consists a single genus, Dictyoglomus, with two type strains and several related pure culture isolates. All isolates are thermophilic anaerobic Gram-type negative rods. A major distinguishing phenotypic feature is the formation of spherical bodies in late stationary phase of growth, the function of which is not understood.Most isolates are fermentative using a range of simple carbohydrates, but some isolates are able to grow on crystalline cellulose and chemolithotrophy using carbon monoxide as energy source has been reported for one pure culture. There have been relatively few applications for Dictyoglomus enzymes as a result of a number of factors. Although many of their kinetic properties are exceptional, they have had to be cloned and expressed in standard fermentation strains as hosts, and their low G:C content has required significant genetic manipulation to provide expression. Some of the main applications have required inexpensive enzymes in bulk (e.g., pulp bleaching in paper manufacture), and they have had to be regarded as a replacement for well-established enzymes currently used in the industry. The major applications have involved glycosyl hydrolases, but new uses in value-added products involving drug precursor transformations have been reported recently.10 page(s