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A study presents a biochemical and structural analysis of [N.sup.5]-carboxyaminoimidazole ribonucleotide ([N.sup.5]-CAIR) mutase (PurE), obtained from the acidophilic bacterium Acetobacter aceti. The results show that PurE facilitates protonation of CAIR C4 in the reverse, nonbiosynthetic direction, as the resulting CAIR is strongly stabilized by the interactions with the enzyme and the water molecule.

Biochemical analysis reveals that the mutant aminoimidazole ribonucleotide synthase protein is defective in ATP binding and this defect can be corrected by high levels of formyl-glycinamidine ribonucleotide (FGAM) in Salmonella enterica. Data indicate that that FGAM compensates for the decreased affinity for ATP by the mutant enzyme.

Research has been conducted on the polyhydroxybutyrate (PHB) synthases. The conversion of the beta-hydroxybutyryl coenzyme A to PHB is described.

Results describe the three-dimensional structures of N(sup)5-carboxyaminoimidazole ribonucleotide synthetase (PurK) associated with ligands at 2.1 and 2.5 angstrom resolution. Crystallographic data indicate that the nature of PurK folding is structurally similar to the ATP grasp superfamily.

Magnetic circular dichroism (MCD) and X-ray absorption (XAS) spectroscopies were utilized to determine the structure of the catalytically relevant ferrous active site of phenylalanine hydroxylase (PAH). MCD and XAS analysis of PAH indicated the presence of a resting ferrous site with a six-coordinate distorted octahedral geometry. The resting catalytically active ferrous site of PAH also contains longer bond distances compared to small molecule complexes.

The tetrahydrobiopterin-dependent aromatic amino acid hydroxylases hydroxylate L-Phe and L-Trp to generate similar oxygenating intermediates. Phenylalanine, tyrosine and tryptophan hydroxylases are involved in the rate-determining steps of phenylalanine catabolism, whose dysfunctioning causes phenylketonuria. A tetramer unit brings about the cooperative unit functioning in the allosteric activation of BH4-dependent catalysis.

A study was carried out to examine all the processes that take place in the condensation half-reaction executed by Thermoplasma acidophilum citrate synthase (TpCS). Findings revealed that the nonhydrolyzable product of the condensation reaction, dethiacitryl-CoA, forms a particularly stable complex with TpCS but not CS.

The crystal structure of acetobacter aceti citrate synthase (AaCS), a hexameric type II citrate synthase, complexed with oxaloacetate and the inhibitor carboxymethyldethia-coenzyme A (CMX), is determined to 1.85-Angstrom resolution employing protein purified by a tandem affinity purification procedure. Its active site residue interaction with oxaloacetate and CMX suggest that AaCS is not regulated by NADH, but it retains many of the residues used by Escherichia coli CS for NADH binding.

The x-ray crystal structure of glycinamide ribonucleotide synthetase (GAR-syn) from Escherichia coli was described. Results revealed that GAR-syn is a member of the ATP-grasp superfamily. Its structure was sterically and electrostatically compatible with the binding surface proposed to be important in amidotransferase (AT). The N domain of GAR-syn was opposite to the flexible loop region of AT that can adopt several conformations and appeared ready to gate phosphoribosylamine release.

Citrate synthase (CS) plays a central metabolic role in aerobes and many other organisms. The CS reaction comprises two half-reactions: a Claisen aldol condensation of acetyl-CoA (AcCoA) and oxaloacetate (OAA) that forms citryl-CoA (CitCoA), and CitCoA hydrolysis. Protein conformational changes that `close' the active site play an important role in the assembly of a catalytically competent condensation active site. CS from the thermoacidophile Thermoplasma acidophilum (TpCS) possesses an endogenous Trp fluorophore that can be used to monitor the condensation reaction. The 2.2 Å resolution crystal structure of TpCS fused to a C-terminal hexahistidine tag (TpCSH6) reported here is an `open' structure that, when compared with several liganded TpCS structures, helps to define a complete path for active-site closure. One active site in each dimer binds a neighboring His tag, the first nonsubstrate ligand known to occupy both the AcCoA and OAA binding sites. Solution data collectively suggest that this fortuitous interaction is stabilized by the crystalline lattice. As a polar but almost neutral ligand, the active site-tail interaction provides a new starting point for the design of bisubstrate-analog inhibitors of CS. [ABSTRACT FROM AUTHOR]

Many food plants accumulate oxalate, which humans absorb but do not metabolize, leading to the formation of urinary stones. The commensal bacterium Oxalobacter formigenes consumes oxalate by converting it to oxalyl-CoA, which is decarboxylated by oxalyl-CoA decarboxylase (OXC). OXC and the class III CoA-transferase formyl-CoA:oxalate CoA-transferase (FCOCT) are widespread among bacteria, including many that have no apparent ability to degrade or to resist external oxalate. The EvgA acid response regulator activates transcription of the Escherichia coli yfdXWUVE operon encoding YfdW (FCOCT), YfdU (OXC), and YfdE, a class III CoA-transferase that is 30% identical to YfdW. YfdW and YfdU are necessary and sufficient for oxalate-induced protection against a subsequent acid challenge; neither of the other genes has a known function. We report the purification, in vitro characterization, 2.1-Å crystal structure, and functional assignment of YfdE. YfdE and UctC, an orthologue from the obligate aerobe Acetobacter aceti, perform the reversible conversion of acetyl-CoA and oxalate to oxalyl-CoA and acetate. The annotation of YfdE as acetyl-CoA:oxalate CoA-transferase (ACOCT) expands the scope of metabolic pathways linked to oxalate catabolism and the oxalate-induced acid tolerance response. FCOCT and ACOCT active sites contain distinctive, conserved active site loops (the glycine-rich loop and the GNxH loop, respectively) that appear to encode substrate specificity. [ABSTRACT FROM AUTHOR]

Coenzyme A (CoA)-transferases catalyze transthioesterification reactions involving acyl-CoA substrates, using an active-site carboxylate to form covalent acyl anhydride and CoA thioester adducts Mechanistic studies of dass I CoA- transferases suggested that acyl-CoA binding energy is used to accelerate rate-linsiting acyl transfers by compressing the substrate thioester tightly against the catalytic glutamate [White, H., and Jencks, W. P. (1976) J. BioL Che,n. 2.51, 1688-1699]. The dass I CoA-transferase succinyl-CoA:acetate CoA-transferase is an acetic acid resistance factor (AarC) with a role in a variant citric acid cyde in Acetobacter aceti. In crystal structures of a C-terminally His6-tagged form (AarCH6) were determined for several wild-type and mutant complexes, induding freeze-trapped acetylglutamyl anhydride and glutamyl-CoA thioester adducts. The latter shows the acetate product bound to an auxiliary Site that is required for efficient carboxylate substrate recognition. A mutant in which the catalytic glutamate was changed to an alanine crystallized in a dosed complex containing dethiaacetyl-CoA, which adopts an unusual curled conformation. A model of the acetyl-CoA Michaelis complex demonstrates the compression anticipated four decades ago by Jencks and reveals that the nudeophilic glutamate is held at a near-ideal angle for attack as the thioester oxygen is forced into an oxyanion hole composed of G1y388 NH and CoA N2″. CoA is nearly immobile along its entire length during all stages of the enzyme reaction. Spatial and sequence conservation of key residues indicates that this mechanism is general among dass I CoA- transferases. [ABSTRACT FROM AUTHOR]

Crepis alpina acetylenase is a variant FAD2 desaturase that catalyses the insertion of a triple bond at the Δ12 position of linoleic acid, forming crepenynic acid in developing seeds. Seeds contain a high level of crepenynic acid but other tissues contain none. Using reverse transcriptase-coupled PCR (RT-PCR), acetylenase transcripts were identified in non-seed C. alpina tissues, which were highest in flower heads. To understand why functional expression of the acetylenase is limited to seeds, genes that affect acetylenase activity by providing substrate (FAD2) or electrons (cytochrome b5), or that compete for substrate (FAD3), were cloned. RT-PCR analysis indicated that the availability of a preferred cytochrome b5 isoform is not a limiting factor. Developing seeds co-express acetylenase and FAD2 isoform 2 (FAD2-2) at high levels. Flower heads co-express FAD2-3 and FAD3 at high levels, and FAD2-2 and acetylenase at moderate levels. FAD2-3 was not expressed in developing seed. Real-time RT-PCR absolute transcript quantitation showed 104-fold higher acetylenase expression in developing seeds than in flower heads. Collectively, the results show that both the acetylenase expression level and the co-expression of other desaturases may contribute to the tissue specificity of crepenynate production. Helianthus annuus contains a Δ12 acetylenase in a polyacetylene biosynthetic pathway, so does not accumulate crepenynate. Real-time RT-PCR analysis showed relatively strong acetylenase expression in young sunflowers. Acetylenase transcription is observed in both species without accumulation of the enzymatic product, crepenynate. Functional expression of acetylenase appears to be affected by competition and collaboration with other enzymes. [ABSTRACT FROM PUBLISHER]

The crystal structure of thioredoxin ( AaTrx) from the acetic acid bacterium Acetobacter aceti was determined at 1 Å resolution. This is currently the highest resolution crystal structure available for any thioredoxin. Thioredoxins facilitate thiol-disulfide exchange, a process that is expected to be slow at the low pH values encountered in the A. aceti cytoplasm. Despite the apparent need to function at low pH, neither the active site nor the surface charge distribution of AaTrx is notably different from that of Escherichia coli thioredoxin. Apparently the ancestral thioredoxin was sufficiently stable for use in A. aceti or the need to interact with multiple targets constrained the variation of surface residues. The AaTrx structure presented here provides a clear view of all ionizable protein moieties and waters, a first step in understanding how thiol-disulfide exchange might occur in a low pH cytoplasm, and is a basis for biophysical studies of the mechanism of acid-mediated unfolding. The high resolution of this structure should be useful for computational studies of thioredoxin function, protein structure and dynamics, and side-chain ionization. [ABSTRACT FROM AUTHOR]

The crystal structure of Acetobacter aceti PurE was determined to a resolution of 1.55 Å and is compared with the known structures of the class I PurEs from a mesophile, Escherichia coli, and a thermophile, Thermotoga maritima. Analyses of the general factors that increase protein stability are examined as potential explanations for the acid stability of A. aceti PurE. Increased inter-subunit hydrogen bonding and an increased number of arginine-containing salt bridges appear to account for the bulk of the increased acid stability. A chain of histidines linking two active sites is discussed in the context of the proton transfers catalyzed by the enzyme. [ABSTRACT FROM AUTHOR]

Presents a mechanistic study of the class III polyhydroxyalkanoate acid (PHA) synthase from Chromatium vinosum. Indication that the C. vinosum PHA synthase is a member of the lipase superfamily; Indication that PhaC and the prokaryotic lipases share a similar fold in their catalytic domain containing the active site nucleophile.

The crystal structure of thioredoxin (AaTrx) from the acetic acid bacterium Acetobacter aceti was determined at 1 A resolution. This is currently the highest resolution crystal structure available for any thioredoxin. Thioredoxins facilitate thiol-disulfide exchange, a process that is expected to be slow at the low pH values encountered in the A. aceti cytoplasm. Despite the apparent need to function at low pH, neither the active site nor the surface charge distribution of AaTrx is notably different from that of Escherichia coli thioredoxin. Apparently the ancestral thioredoxin was sufficiently stable for use in A. aceti or the need to interact with multiple targets constrained the variation of surface residues. The AaTrx structure presented here provides a clear view of all ionizable protein moieties and waters, a first step in understanding how thiol-disulfide exchange might occur in a low pH cytoplasm, and is a basis for biophysical studies of the mechanism of acid-mediated unfolding. The high resolution of this structure should be useful for computational studies of thioredoxin function, protein structure and dynamics, and side-chain ionization.

The crystal structure of Acetobacter aceti PurE was determined to a resolution of 1.55 A and is compared with the known structures of the class I PurEs from a mesophile, Escherichia coli, and a thermophile, Thermotoga maritima. Analyses of the general factors that increase protein stability are examined as potential explanations for the acid stability of A. aceti PurE. Increased inter-subunit hydrogen bonding and an increased number of arginine-containing salt bridges appear to account for the bulk of the increased acid stability. A chain of histidines linking two active sites is discussed in the context of the proton transfers catalyzed by the enzyme.