Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to the corresponding deoxyribonucleotides, used in DNA synthesis and repair. Two different mechanisms help deliver the required electrons to the RNR active site. Formate can be used as reductant directly in the active site, or glutaredoxins or thioredoxins reduce a C-terminal cysteine pair, which then delivers the electrons to the active site. Here, we characterized a novel cysteine-rich C-terminal domain (CRD), which is present in most class II RNRs found in microbes. The NrdJd-type RNR from the bacterium Stackebrandtia nassauensis was used as a model enzyme. We show that the CRD is involved in both higher oligomeric state formation and electron transfer to the active site. The CRD-dependent formation of high oligomers, such as tetramers and hexamers, was induced by addition of dATP or dGTP, but not of dTTP or dCTP. The electron transfer was mediated by an array of six cysteine residues at the very C-terminal end, which also coordinated a zinc atom. The electron transfer can also occur between subunits, depending on the enzyme's oligomeric state. An investigation of the native reductant of the system revealed no interaction with glutaredoxins or thioredoxins, indicating that this class II RNR uses a different electron source. Our results indicate that the CRD has a crucial role in catalytic turnover and a potentially new terminal reduction mechanism and suggest that the CRD is important for the activities of many class II RNRs.

Summary At the end of the cell cycle, the plant cell wall is deposited within a membrane compartment referred to as the cell plate. Little is known about the biogenesis of this transient membrane compartment. We have positionally cloned and characterized a novel Arabidopsis gene, CLUB, identified by mutation. CLUB/AtTRS130 encodes a putative TRAPPII tethering factor. club mutants are seedling‐lethal and have a canonical cytokinesis‐defective phenotype, characterized by the appearance of bi‐ or multinucleate cells with cell wall stubs, gaps and floating walls. Confocal microscopy showed that in club mutants, KNOLLE‐positive vesicles formed and accumulated at the cell equator throughout cytokinesis, but failed to assemble into a cell plate. Similarly, electron micrographs showed large vesicles loosely connected as patchy, incomplete cell plates in club root tips. Neither the formation of KNOLLE‐positive vesicles nor the delivery of these vesicles to the cell equator appeared to be perturbed in club mutants. Thus, the primary defect in club mutants appears to be an impairment in cell plate assembly. As a putative tethering factor required for cell plate biogenesis, CLUB/AtTRS130 helps to define the identity of this membrane compartment and comprises an important handle on the regulation of cell plate assembly.

The (S)‐selective carbonyl reductase CPCR2 from Candida parapsilosis is a member of the medium‐chain reductase family of enzymes and is a useful biocatalyst for the reduction of prochiral ketone substrates. The structure of CPCR2 was determined in complex with the cofactor NADH [NADH=reduced form of nicotinamide adenine dinucleotide (NAD+)] to a resolution of 2.05 Å. Two dimers formed a tetramer in the asymmetric unit, but solution studies confirmed that a dimer was the predominant species in solution. In the monomer, the NADH cofactor is bound at the interface between the nucleotide binding domain and the catalytic domain, and the Re‐face hydride of the nicotinamide ring is presented to a hydrophobic binding pocket featuring the Leu262, Phe285, Trp286, Trp116, Leu119, Leu55 and Val50 residues, which leads to the surface of the enzyme. The catalytic zinc and coordinating amino acid side chains were observed in different conformations in the different monomers. In three out of four monomers, the zinc was coordinated by His65, Asp154, Glu66 and a water molecule; in the other subunit, an alternative coordination sphere, consisting of His65, Asp154, Cys44 and a water molecule, was observed. The change in coordination was accompanied by a movement of a mobile region of the protein chain between residues 43 and 63, which bears Cys44. The structure of CPCR2 provides further evidence of a dynamic coordination sphere for zinc in medium‐chain reductase dependent catalysis. It also sheds light on previous engineering studies on CPCR2 that were performed in the absence of structural data and provides a robust and reliable new model for further experiments directed towards improvement or alteration of CPCR2 activity.

Molecular docking of substrates is more challenging compared to inhibitors as the reaction mechanism has to be considered. This becomes more pronounced for zinc-dependent enzymes since the coordination state of the catalytic zinc ion is of greater importance. In order to develop a predictive substrate docking protocol, we have performed molecular docking studies of diketone substrates using the catalytic state of carbonyl reductase 2 from Candida parapsilosis (CPCR2). Different docking protocols using two docking methods (AutoDock Vina and AutoDock4.2) with two different sets of atomic charges (AM1-BCC and HF-RESP) for catalytic zinc environment and substrates as well as two sets of vdW parameters for zinc ion were examined. We have selected the catalytic binding pose of each substrate by applying mechanism based distance criteria. To compare the performance of the docking protocols, the correlation plots for the binding energies of these catalytic poses were obtained against experimental Vmax values of the 11 diketone substrates for CPCR2. The best correlation of 0.73 was achieved with AutoDock4.2 while treating catalytic zinc ion in optimized non-bonded (NBopt) state with +1.01 charge on the zinc ion, compared to 0.36 in non-bonded (+2.00 charge on the zinc ion) state. These results indicate the importance of catalytic constraints and charge parameterization of catalytic zinc environment for the prediction of substrate activity in zinc-dependent enzymes by molecular docking. The developed predictive docking protocol described here is in principle generally applicable for the efficient in silico substrate spectra characterization of zinc-dependent ADH.

Zinc-dependent alcohol dehydrogenases (ADHs) are valuable biocatalystsf or the synthesis of chiral hydroxy compoundss uch as a-hydroxy ketones andd iols, both valuable precursors for the synthesis of various pharmaceuticals.H owever, while highly active on aliphatic or phenyl-substituted diketones,m ost well characterized ADHs show no significant activity on cyclic a-a nd b-diketones.T herefore, this study aimed at the detection of anovel ADHcapable to reduce these special targets.Itinvolved arational screeningo fb iochemical pathways for enzymes with structurally related natural substrates. Thes od etected 6-hydroxycyclohex-1-ene-1-carbonyl-CoA dehydrogenase (ThaADH) from Thauera aromatica was cloned, expressedi nEscherichiac oli and purified by affinity chromatography.T he characterization revealed as ubstrate specificity with highest activitieso nc yclic a-a nd b-diketones including 1,2-cyclohexanedionea nd 1,3-cyclopentanedione. Structural reasonsf or this extraordinary substrate spectrumw ere investigated with ah omology model created via Swiss Model server. Although the quality of the model may be improved, it suggestst hat ab ulky aromaticr esidue,t hat plays ac rucial role in the definition of the substrate binding pockets of most ADHs,i sr eplaced by ag lycine residue in ThaADH. We propose that this structural difference leads to the formation of one large binding pocket insteado ft wo smallero nes and consequently to ap reference for cyclic diketones over linear bulky substrates.T hus,w eh ave achieved both provision of an ovelb iocatalyst with high potential in chiral synthesis,a nd ap ossible explanation for the measured differencest ok nown ADHs.T he described structural motif might be usedf or identification of further enzymeswith ar elated substrate scope.