NF-κB essential modulator (NEMO) interaction with linear and lys-63 ubiquitin chains contributes to NF-κB activation.

The IκB kinase (IKK) complex acts as a gatekeeper of canonical NF-κB signaling in response to upstream stimulation. IKK activation requires sensing of ubiquitin chains by the essential IKK regulatory subunit IKKγ/NEMO. However, it has remained enigmatic whether NEMO binding to Lys-63-linked or linear ubiquitin chains is critical for triggering IKK activation. We show here that the NEMO C terminus, comprising the ubiquitin binding region and a zinc finger, has a high preference for binding to linear ubiquitin chains. However, immobilization of NEMO, which may be reminiscent of cellular oligomerization, facilitates the interaction with Lys-63 ubiquitin chains. Moreover, selective mutations in NEMO that abolish association with linear ubiquitin but do not affect binding to Lys-63 ubiquitin are only partially compromising NF-κB signaling in response to TNFα stimulation in fibroblasts and T cells. In line with this, TNFα-triggered expression of NF-κB target genes and induction of apoptosis was partially compromised by NEMO mutations that selectively impair the binding to linear ubiquitin chains. Thus, in vivo NEMO interaction with linear and Lys-63 ubiquitin chains is required for optimal IKK activation, suggesting that both type of chains are cooperating in triggering canonical NF-κB signaling.

Structural studies of mutant forms of the PQQ-forming enzyme PqqC in the presence of product and substrate.

Pyrroloquinoline quinone [4,5-dihydro-4,5-dioxo-1H-pyrrolo[2,3-f]quinoline-2,7,9-tricarboxylic acid (PQQ)] is a bacterial cofactor in numerous alcohol dehydrogenases including methanol dehydrogenase and glucose dehydrogenase. Its biosynthesis in Klebsiella pneumoniae is facilitated by six genes, pqqABCDEF and proceeds by an unknown pathway. PqqC is one of two metal free oxidases of known structure and catalyzes the last step of PQQ biogenesis which involves a ring closure and an eight-electron oxidation of the substrate [3a-(2-amino-2-carboxyethyl)-4,5-dioxo-4,5,6,7,8,9-hexahydroquinoline-7,9-dicarboxylic acid (AHQQ)]. PqqC has 14 conserved active site residues, which have previously been shown to be in close contact with bound PQQ. Herein, we describe the structures of three PqqC active site variants, H154S, Y175F, and the double mutant R179S/Y175S. The H154S crystal structure shows that, even with PQQ bound, the enzyme is still in the "open" conformation with helices alpha5b and alpha6 unfolded and the active site solvent accessible. The Y175F PQQ complex crystal structure reveals the closed conformation indicating that Y175 is not required for the conformational change. The R179S/Y175S AHQQ complex crystal structure is the most mechanistically informative, indicating an open conformation with a reaction intermediate trapped in the active site. The intermediate seen in R179S/Y175S is tricyclic but nonplanar, implying that it has not undergone oxidation. These studies implicate a stepwise process in which substrate binding leads to the generation of the closed protein conformation, with the latter playing a critical role in O(2) binding and catalysis.

The pyrroloquinoline quinone biosynthesis pathway revisited: a structural approach.

BACKGROUND: The biosynthesis pathway of Pyrroloquinoline quinone, a bacterial redox active cofactor for numerous alcohol and aldose dehydrogenases, is largely unknown, but it is proven that at least six genes in Klebsiella pneumoniae (PqqA-F) are required, all of which are located in the PQQ-operon. RESULTS: New structural data of some PQQ biosynthesis proteins and their homologues provide new insights and functional assignments of the proteins in the pathway. Based on sequence analysis and homology models we propose the role and catalytic function for each enzyme involved in this intriguing biosynthesis pathway. CONCLUSION: PQQ is derived from the two amino acids glutamate and tyrosine encoded in the precursor peptide PqqA. Five reactions are necessary to form this quinone cofactor. The PqqA peptide is recognised by PqqE, which links the C9 and C9a, afterwards it is accepted by PqqF which cuts out the linked amino acids. The next reaction (Schiff base) is spontaneous, the following dioxygenation is catalysed by an unknown enzyme. The last cyclization and oxidation steps are catalysed by PqqC. Taken together the known facts of the different proteins we assign a putative function to all six proteins in PQQ biosynthesis pathway.