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 (K M 0.06 mM at pH 5), high catalytic efficiencies (1.3 • 10(6) M(-1) • s(-1) at pH 5), pHopt of 5.5 and Topt at 45°C. The enzyme is not thermostable (T½ of 18 minutes at 60°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 Å for Cα 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.

The shape-shifting quasispecies of RNA: one sequence, many functional folds.

E Unus pluribum, or "Of One, Many", may be at the root of decoding the RNA sequence-structure-function relationship. RNAs embody the large majority of genes in higher eukaryotes and fold in a sequence-directed fashion into three-dimensional structures that perform functions conserved across all cellular life forms, ranging from regulating to executing gene expression. While it is the most important determinant of the RNA structure, the nucleotide sequence is generally not sufficient to specify a unique set of secondary and tertiary interactions due to the highly frustrated nature of RNA folding. This frustration results in folding heterogeneity, a common phenomenon wherein a chemically homogeneous population of RNA molecules folds into multiple stable structures. Often, these alternative conformations constitute misfolds, lacking the biological activity of the natively folded RNA. Intriguingly, a number of RNAs have recently been described as capable of adopting multiple distinct conformations that all perform, or contribute to, the same function. Characteristically, these conformations interconvert slowly on the experimental timescale, suggesting that they should be regarded as distinct native states. We discuss how rugged folding free energy landscapes give rise to multiple native states in the Tetrahymena Group I intron ribozyme, hairpin ribozyme, sarcin-ricin loop, ribosome, and an in vitro selected aptamer. We further describe the varying degrees to which folding heterogeneity impacts function in these RNAs, and compare and contrast this impact with that of heterogeneities found in protein folding. Embracing that one sequence can give rise to multiple native folds, we hypothesize that this phenomenon imparts adaptive advantages on any functionally evolving RNA quasispecies.

Oligosaccharide beta-glucans with unusual linkages from Sarcina ventriculi.

The structure of a family of unusual glucans from Sarcina ventriculi has been characterized by NMR spectroscopy, methylation analysis, and mass spectrometry. One is a trisaccharide containing a beta-(1-->3) and a beta-(1-->4)-linkage. The other is a hexasaccharide that is simply a 1,4-linkage dimer of the trisaccharide unit. This is the first report of beta-glucan biosynthesis in a Gram-positive organism. Their occurrence in these organisms supports an even more general link between their synthesis and the adaptability of bacteria.

Membrane lipid alkyl chain motional dynamics is conserved in Sarcina ventriculi despite pH-induced adaptative structural modifications including alkyl chain tail to tail coupling.

Sarcina ventriculi, an anaerobic Gram-positive bacterium, adapts to increasing temperature, the presence of organic solvents, or the lowering of the pH of its growth medium by joining the tails of membrane lipids from opposite sides of the bilayer, forming transmembrane, bifunctional fatty acid species. Since this is done to offset the increase in membrane mobility caused by these perturbations, it is of interest to determine whether the motional (dynamic) properties of membrane lipid alkyl chains are conserved. In this study, conservation of the motional time scales of the alkyl chains of total membrane lipids from Sarcina ventriculi cells grown at different pH values was demonstrated using proton nuclear magnetic resonance (NMR) spectroscopy. The NMR longitudinal relaxation times (T1) of the protons in the bulk methylene groups were measured for lipids from cells grown at pH 3.0 and 7.0. These measurements indicated that the temperature profile of the T1 relaxation behavior for the methylene protons from these two different preparations was the same. Analysis of the data from T1 measurements indicated that the thermal barrier for relaxation is the same in both lipid systems. This is only true if the pH of the sample on which the measurement is being made is adjusted to the same value as that at which the corresponding cells were cultured. It is clear from this latter observation that the state of protonation of the lipid head groups is a contributor to the overall motional freedom of the membrane lipid components. The correlation times (tau c) of characteristic lipid alkyl chain motion were estimated to be approximately 10(-10) s.(ABSTRACT TRUNCATED AT 250 WORDS)

Structures and stereochemistry of the very long alpha, omega-bifunctional alkyl species in the membrane of Sarcina ventriculi indicate that they are formed by tail-to-tail coupling of normal fatty acids.

In a previous study, we demonstrated that Sarcina ventriculi is capable of adjusting to alterations in environmental conditions (such as increase in temperature, lowering of pH, or addition of exogenous organic solvents) by the synthesis of a family of alpha, omega-dicarboxylic acids ranging from 28 to 36 carbons long (Jung, S., et al. 1993. J. Biol. Chem. 268: 2828-2835). The chain lengths and relative abundance of the very long dicarboxylic acids found in S. ventriculi suggest that they may be formed after the perturbation by the (enzymatic) tail-to-tail combinations of existing regular monofunctional fatty acids and not completely de novo by direct 2-carbon addition of acetyl coenzyme A (CoA). If this were true, knowing the structures of the regular fatty acids, we can predict those of the very long chain bifunctional acids. In this work we present definitive chemical results that strongly support this mechanism. This was done by analyzing the structures and stereochemistry of the very long bifunctional species in the light of those of the regular monofunctional species. The exact structures of membrane fatty acid methyl ester derivatives components were determined by various spectroscopic and chemical methods including gas chromatographic (GC) analysis, gas chromatography-mass spectrometry (GC-MS), 1H and 13C nuclear magnetic resonance (NMR) spectroscopy, Fourier transform infrared (FTIR) spectroscopy, polarimetry, and reductive ozonolysis. This yielded precise structural and stereochemical information on the position of substitution of the acyl chain by methyl groups, position and configuration of double bonds, and optical activity. These results, coupled with the absence of intermediate length acyl species, indicated that the very long alkyl species (without exception) can be formed by tail-to-tail joining of existing fatty acids. The ideas of a dynamically regulated catalytic system is proposed.