A dynamic C-terminal segment in the Mycobacterium tuberculosis Mn/Fe R2lox protein can adopt a helical structure with possible functional consequences.

Mycobacterium tuberculosis R2-like ligand-binding oxidase (MtR2lox) belongs to a recently discovered group of proteins that are homologous to the ribonucleotide reductase R2 proteins. MtR2lox carries a heterodinuclear Mn/Fe cofactor and, unlike R2 proteins, a large ligand-binding cavity. A unique tyrosine-valine cross link is also found in the vicinity of the active site.

Proton-transport mechanisms in cytochrome c oxidase revealed by studies of kinetic isotope effects.

Cytochrome c oxidase (CytcO) is a membrane-bound enzyme, which catalyzes the reduction of di-oxygen to water and uses a major part of the free energy released in this reaction to pump protons across the membrane. In the Rhodobacter sphaeroides aa₃ CytcO all protons that are pumped across the membrane, as well as one half of the protons that are used for O₂ reduction, are transferred through one specific intraprotein proton pathway, which holds a highly conserved Glu286 residue. Key questions that need to be addressed in order to understand the function of CytcO at a molecular level are related to the timing of proton transfers from Glu286 to a "pump site" and the catalytic site, respectively.

Mechanism of ADP-ribosylation removal revealed by the structure and ligand complexes of the dimanganese mono-ADP-ribosylhydrolase DraG.

ADP-ribosylation is a ubiquitous regulatory posttranslational modification involved in numerous key processes such as DNA repair, transcription, cell differentiation, apoptosis, and the pathogenic mechanism of certain bacterial toxins. Despite the importance of this reversible process, very little is known about the structure and mechanism of the hydrolases that catalyze removal of the ADP-ribose moiety. In the phototrophic bacterium Rhodospirillum rubrum, dinitrogenase reductase-activating glycohydrolase (DraG), a dimanganese enzyme that reversibly associates with the cell membrane, is a key player in the regulation of nitrogenase activity.

Three-dimensional structures of apo- and holo-L-alanine dehydrogenase from Mycobacterium tuberculosis reveal conformational changes upon coenzyme binding.

L-alanine dehydrogenase from Mycobacterium tuberculosis catalyzes the NADH-dependent reversible conversion of pyruvate and ammonia to L-alanine. Expression of the gene coding for this enzyme is up-regulated in the persistent phase of the organism, and alanine dehydrogenase is therefore a potential target for pathogen control by antibacterial compounds. We have determined the crystal structures of the apo- and holo-forms of the enzyme to 2.

Differential substrate specificity and kinetic behavior of Escherichia coli YfdW and Oxalobacter formigenes formyl coenzyme A transferase.

The yfdXWUVE operon appears to encode proteins that enhance the ability of Escherichia coli MG1655 to survive under acidic conditions. Although the molecular mechanisms underlying this phenotypic behavior remain to be elucidated, findings from structural genomic studies have shown that the structure of YfdW, the protein encoded by the yfdW gene, is homologous to that of the enzyme that mediates oxalate catabolism in the obligate anaerobe Oxalobacter formigenes, O. formigenes formyl coenzyme A transferase (FRC).

Rational protein design of ThDP-dependent enzymes-engineering stereoselectivity.

Benzoylformate decarboxylase (BFD) from Pseudomonas putida is an exceptional thiamin diphosphate-dependent enzyme, as it catalyzes the formation of (S)-2-hydroxy-1-phenylpropan-1-one from benzaldehyde and acetaldehyde. This is the only currently known S-selective reaction (92 % ee) catalyzed by this otherwise R-selective class of enzymes. Here we describe the molecular basis of the introduction of S selectivity into ThDP-dependent decarboxylases.

Reinvestigation of the catalytic mechanism of formyl-CoA transferase, a class III CoA-transferase.

Formyl-coenzyme A transferase from Oxalobacter formigenes belongs to the Class III coenzyme A transferase family and catalyzes the reversible transfer of a CoA carrier between formyl-CoA and oxalate, forming oxalyl-CoA and formate. Formyl-CoA transferase has a unique three-dimensional fold composed of two interlaced subunits locked together like rings of a chain. We here present an intermediate in the reaction, formyl-CoA transferase containing the covalent beta-aspartyl-CoA thioester, adopting different conformations in the two active sites of the dimer, which was identified through crystallographic freeze-trapping experiments with formyl-CoA and oxalyl-CoA in the absence of acceptor carboxylic acid.

Structure of the branched-chain keto acid decarboxylase (KdcA) from Lactococcus lactis provides insights into the structural basis for the chemoselective and enantioselective carboligation reaction.

The thiamin diphosphate (ThDP) dependent branched-chain keto acid decarboxylase (KdcA) from Lactococcus lactis catalyzes the decarboxylation of 3-methyl-2-oxobutanoic acid to 3-methylpropanal (isobutyraldehyde) and CO2. The enzyme is also able to catalyze carboligation reactions with an exceptionally broad substrate range, a feature that makes KdcA a potentially valuable biocatalyst for C-C bond formation, in particular for the enzymatic synthesis of diversely substituted 2-hydroxyketones with high enantioselectivity. The crystal structures of recombinant holo-KdcA and of a complex with an inhibitory ThDP analogue mimicking a reaction intermediate have been determined to resolutions of 1.

Crystallographic snapshots of oxalyl-CoA decarboxylase give insights into catalysis by nonoxidative ThDP-dependent decarboxylases.

Despite more than five decades of extensive studies of thiamin diphosphate (ThDP) enzymes, there remain many uncertainties as to how these enzymes achieve their rate enhancements. Here, we present a clear picture of catalysis for the simple nonoxidative decarboxylase, oxalyl-coenzyme A (CoA) decarboxylase, based on crystallographic snapshots along the catalytic cycle and kinetic data on active site mutants. First, we provide crystallographic evidence that, upon binding of oxalyl-CoA, the C-terminal 13 residues fold over the substrate, aligning the substrate alpha-carbon for attack by the ThDP-C2 atom.

Structural basis for activation of the thiamin diphosphate-dependent enzyme oxalyl-CoA decarboxylase by adenosine diphosphate.

Oxalyl-coenzyme A decarboxylase is a thiamin diphosphate-dependent enzyme that plays an important role in the catabolism of the highly toxic compound oxalate. We have determined the crystal structure of the enzyme from Oxalobacter formigenes from a hemihedrally twinned crystal to 1.73 A resolution and characterized the steady-state kinetic behavior of the decarboxylase.

Detection and characterization of merohedral twinning in crystals of oxalyl-coenzyme A decarboxylase from Oxalobacter formigenes.

Oxalyl-coenzyme A decarboxylase is a thiamin diphosphate dependent enzyme active in the catabolism of the highly toxic compound oxalate. The enzyme from Oxalobacter formigenes has been expressed as a recombinant protein in Escherichia coli, purified to homogeneity and crystallized. Two crystal forms were obtained, one showing poor diffraction and the other merohedral twinning.