To investigate the flavin utilization by dibenzothiophene monooxygenase (DszC), DszC of a desulfurizing bacterium Mycobacterium goodii X7B was purified from the recombinant Escherichia coli. It was shown to be able to utilize either FMNH2 or FADH2 when coupled with a flavin reductase that reduces either FMN or FAD. Sequence analysis indicated that DszC was similar to the C2 component of p-hydroxyphenylacetate hydroxylase from Acinetobacter baumannii, which can use both FADH2 and FMNH2 as substrates. Both flavins at high concentrations could inhibit the activity of DszC due to autocatalytic oxidation of reduced flavins. The results suggest that DszC should be reclassified as an FMNH2 and FADH2 both-utilizing monooxygenase component and the flavins should be controlled at properly reduced levels to obtain optimal biodesulfurization results.

The SCF ubiquitin ligases catalyze protein ubiquitination in diverse cellular processes. SCFs bind substrates through the interchangeable F box protein subunit, with the 70 human F box proteins allowing the recognition of a wide range of substrates. The F box protein β-TrCP1 recognizes the doubly phosphorylated DpSGφXpS destruction motif, present in β-catenin and I B, and directs the SCF β-TrCP1 to ubiquitinate these proteins at specific lysines. The 3.0 A structure of a β-TrCP1-Skp1-β-catenin complex reveals the basis of substrate recognition by the β-TrCP1 WD40 domain. The structure, togetherwith the previous SCFSkp2 structure, leads to the model of SCF catalyzing ubiquitination by increasing the effective concentration of the substrate lysine at the E2 active site. The model's prediction that the lysine-destruction motif spacing is a determinant of ubiquitination efficiency is confirmed by measuring ubiquitination rates of mutant β-catenin peptides, solidifying the model and also providing a mechanistic basis for lysine selection.

The ubiquitin-mediated proteolysis of the Cdk2 inhibitor p27Kip1 plays a central role in cell cycle progression, and enhanced degradation of p27Kip1 is associated with many common cancers. Proteolysis of p27Kip1 is triggered by Thr187 phosphorylation, which leads to the binding of the SCFSkp2 (Skp1-Cul1-Rbx1-Skp2) ubiquitin ligase complex. Unlike other known SCF substrates, p27Kip1 ubiquitination also requires the accessory protein Cks1. The crystal structure of the Skp1-Skp2-Cks1 complex bound to a p27Kip1 phosphopeptide shows that Cks1 binds to the leucine-rich repeat (LRR) domain and C-terminal tail of Skp2, whereas p27Kip1 binds to both Cks1 and Skp2. The phosphorylated Thr187 side chain of p27Kip1 is recognized by a Cks1 phosphate binding site, whereas the side chain of an invariant Glu185 inserts into the interface between Skp2 and Cks1, interacting with both. The structure and biochemical data support the proposed model that Cdk2-cyclin A contributes to the recruitment of p27Kip1 to the SCFSkp2-Cks1 complex.

In the February issue of Developmental Cell, Nastasi et al. describe Ozz, a muscle-specific ubiquitin ligase adaptor that regulates myofibril organization. Ozz appears to function in ubiquitination and degradation of membrane-bound, but not eytosolie, β-eatenin,whose turnover may be required for alignment and growth of the sareomere, the basic contractile unit of myofibers.

β-Catenin phosphorylation at serine 45 (Ser45), threonine 41 (Thr41), Ser37, and Ser33 is critical for β-catenin degradation, and regulation of β-catenin phosphorylation is a central part of the canonical Wnt signaling pathway. β-Catenin mutations at Ser45, Thr41, Ser37, and Ser33 perturb β-catenin degradation and are frequently found in cancers. It is established that Ser45 phosphorylation by casein kinase I (CKI) initiates phosphorylation at Thr41, Ser37, and Ser33 by glycogen synthase kinase 3 (GSK3) and that phosphorylated Ser37 and Ser33 are recognized by the F-box protein β-TrCP, a component of a ubiquitin ligase complex that mediates β-catenin degradation. While the roles of Ser45, Ser37, and Ser33 are well documented, the function of Thr41 remains less defined. Here we show that Thr41 strictly acts as a phosphorylation relay residue and that the Ser-X-X-X-Ser (X is any amino acid) motif is obligatory for β-catenin phosphorylation by GSK3. β-Catenin phosphorylation/degradation and its regulation by Wnt can occur normally in the absence of Thr41 as long as the Ser-X-X-X-Ser motif/spacing is preserved. These results suggest that Thr41 functions to bridge sequential phosphorylation from Ser45 to Ser37 and provide further insights into the discrete steps and logic in β-catenin phosphorylation-degradation.