Inactive pseudoenzyme subunits in heterotetrameric BbsCD, a novel short-chain alcohol dehydrogenase involved in anaerobic toluene degradation.

Anaerobic toluene degradation proceeds by fumarate addition to produce (R)-benzylsuccinate as first intermediate, which is further degraded via ß-oxidation by five enzymes encoded in the conserved bbs operon. This study characterizes two enzymes of this pathway, (E)-benzylidenesuccinyl-CoA hydratase (BbsH), and (S,R)-2-(α-hydroxybenzyl)succinyl-CoA dehydrogenase (BbsCD) from Thauera aromatica. BbsH, a member of the enoyl-CoA hydratase family, converts (E)-benzylidenesuccinyl-CoA to 2-(α-hydroxybenzyl)succinyl-CoA and was subsequently used in a coupled enzyme assay with BbsCD, which belongs to the short-chain dehydrogenases/reductase (SDR) family. The BbsCD crystal structure shows a C2-symmetric heterotetramer consisting of BbsC2 and BbsD2 dimers. BbsD subunits are catalytically active and capable of binding NAD+ and substrate, whereas BbsC subunits represent built-in pseudoenzyme moieties lacking all motifs of the SDR family required for substrate binding or catalysis. Molecular modeling studies predict that the active site of BbsD is specific for conversion of the (S,R)-diastereomer of 2-(α-hydroxybenzyl)succinyl-CoA to (S)-2-benzoylsuccinyl-CoA by hydride transfer to the re-face of nicotinamide adenine dinucleotide (NAD)+ . Furthermore, BbsC subunits are not engaged in substrate binding and merely serve as scaffold for the BbsD dimer. BbsCD represents a novel clade of related enzymes within the SDR family, which adopt a heterotetrameric architecture and catalyze the ß-oxidation of aromatic succinate adducts.

Nicotinamide Adenine Dinucleotides Arrest Photoreduction of Class II DNA Photolyases in FADH˙ State.

All light-sensitive members of the photolyase/cryptochrome family rely on FAD as catalytic cofactor. Its activity is regulated by photoreduction, a light-triggered electron transfer process from a conserved tryptophan triad to the flavin. The stability of the reduced flavin depends on available external electron donors and oxygen. In this study, we show for the class II photolyase of Methanosarcina mazei, MmCPDII, that it utilizes physiologically relevant redox cofactors NADH and NADPH for the formation of the semiquinoid FAD in a light-dependent reaction. Using redox-inert variants MmCPDII/W388F and MmCPDII/W360F, we demonstrate that photoreduction by NADH and NADPH requires the class II-specific tryptophan cascade of MmCPDII. Finally, we confirmed that mutations in the tryptophan cascade can be introduced without any substantial structural disturbances by analyzing crystal structures of MmCPDII/W388F, MmCPDII/W360F and MmCPDII/Y345F.

Bioactive cyclometalated phthalimides: design, synthesis and kinase inhibition.

The regioselective cyclometalation of 4-(pyridin-2-yl)phthalimide was exploited for the economical design of organometallic protein kinase inhibitors. 4-(Pyridin-2-yl)phthalimide can be prepared from inexpensive 4-bromophthalimide in just three steps including one Pd-catalyzed Stille cross-coupling. The versatility of this new ligand was demonstrated with the synthesis of ruthenium(II) half-sandwich as well as octahedral ruthenium(II) and iridium(III) complexes. The regioselectivity of the C-H activation in the course of the cyclometalation can be influenced by the reaction conditions and the steric demand of the introduced metal complex fragment. The biological activity of this new class of metalated phthalimides was evaluated by profiling two representative members against a large panel of human protein kinases. A cocrystal structure of one metallo-phthalimide with the protein kinase Pim1 confirmed an ATP-competitive binding with the intended hydrogen bonding between the phthalimide moiety and the hinge region of the ATP-binding site.

CryB from Rhodobacter sphaeroides: a unique class of cryptochromes with new cofactors.

Cryptochromes and photolyases are structurally related but have different biological functions in signalling and DNA repair. Proteobacteria and cyanobacteria harbour a new class of cryptochromes, called CryPro. We have solved the 2.7 Å structure of one of its members, cryptochrome B from Rhodobacter sphaeroides, which is a regulator of photosynthesis gene expression. The structure reveals that, in addition to the photolyase-like fold, CryB contains two cofactors only conserved in the CryPro subfamily: 6,7-dimethyl-8-ribityl-lumazine in the antenna-binding domain and a [4Fe-4S] cluster within the catalytic domain. The latter closely resembles the iron-sulphur cluster harbouring the large primase subunit PriL, indicating that PriL is evolutionarily related to the CryPro class of cryptochromes.

Crystal structures of an archaeal class II DNA photolyase and its complex with UV-damaged duplex DNA.

Class II photolyases ubiquitously occur in plants, animals, prokaryotes and some viruses. Like the distantly related microbial class I photolyases, these enzymes repair UV-induced cyclobutane pyrimidine dimer (CPD) lesions within duplex DNA using blue/near-UV light. Methanosarcina mazei Mm0852 is a class II photolyase of the archaeal order of Methanosarcinales, and is closely related to plant and metazoan counterparts. Mm0852 catalyses light-driven DNA repair and photoreduction, but in contrast to class I enzymes lacks a high degree of binding discrimination between UV-damaged and intact duplex DNA. We solved crystal structures of Mm0852, the first one for a class II photolyase, alone and in complex with CPD lesion-containing duplex DNA. The lesion-binding mode differs from other photolyases by a larger DNA-binding site, and an unrepaired CPD lesion is found flipped into the active site and recognized by a cluster of five water molecules next to the bound 3'-thymine base. Different from other members of the photolyase-cryptochrome family, class II photolyases appear to utilize an unusual, conserved tryptophane dyad as electron transfer pathway to the catalytic FAD cofactor.

Structurally sophisticated octahedral metal complexes as highly selective protein kinase inhibitors.

The generation of synthetic compounds with exclusive target specificity is an extraordinary challenge of molecular recognition and demands novel design strategies, in particular for large and homologous protein families such as protein kinases with more than 500 members. Simple organic molecules often do not reach the necessary sophistication to fulfill this task. Here, we present six carefully tailored, stable metal-containing compounds in which unique and defined molecular geometries with natural-product-like structural complexity are constructed around octahedral ruthenium(II) or iridium(III) metal centers. Each of the six reported metal compounds displays high selectivity for an individual protein kinase, namely GSK3α, PAK1, PIM1, DAPK1, MLCK, and FLT4. Although being conventional ATP-competitive inhibitors, the combination of the unusual globular shape and rigidity characteristics, of these compounds facilitates the design of highly selective protein kinase inhibitors. Unique structural features of the octahedral coordination geometry allow novel interactions with the glycine-rich loop, which contribute significantly to binding potencies and selectivities. The sensitive correlation between metal coordination sphere and inhibition properties suggests that in this design, the metal is located at a "hot spot" within the ATP binding pocket, not too close to the hinge region where globular space is unavailable, and at the same time not too far out toward the solvent where the octahedral coordination sphere would not have a significant impact on potency and selectivity. This study thus demonstrates that inert (stable) octahedral metal complexes are sophisticated structural scaffolds for the design of highly selective chemical probes.

Photoreduction of the folate cofactor in members of the photolyase family.

Cryptochromes and DNA photolyases are related flavoproteins with flavin adenine dinucleotide as the common cofactor. Whereas photolyases repair DNA lesions caused by UV radiation, cryptochromes generally lack repair activity but act as UV-A/blue light photoreceptors. Two distinct electron transfer (ET) pathways have been identified in DNA photolyases. One pathway uses within its catalytic cycle, light-driven electron transfer from FADH(-)* to the DNA lesion and electron back-transfer to semireduced FADH(o) after photoproduct cleavage. This cyclic ET pathway seems to be unique for the photolyase subfamily. The second ET pathway mediates photoreduction of semireduced or fully oxidized FAD via a triad of aromatic residues that is conserved in photolyases and cryptochromes. The 5,10-methenyltetrahydrofolate (5,10-methenylTHF) antenna cofactor in members of the photolyase family is bleached upon light excitation. This process has been described as photodecomposition of 5,10-methenylTHF. We show that photobleaching of 5,10-methenylTHF in Arabidopsis cry3, a member of the cryptochrome DASH family, with repair activity for cyclobutane pyrimidine dimer lesions in single-stranded DNA and in Escherichia coli photolyase results from reduction of 5,10-methenylTHF to 5,10-methyleneTHF that requires the intact tryptophan triad. Thus, a third ET pathway exists in members of the photolyase family that remained undiscovered so far.