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Escherichia coli thioesterase I (TAP) is a multifunctional enzyme possessing activities of thioesterase, esterase, arylesterase, protease, and lysophospholipase. In particular, TAP has stereoselectivity for amino acid derivative substrates, hence it is useful for the kinetic resolution of racemic mixtures of industrial chemicals. In the present work, the crystal structure of native TAP was determined at 1.9 A˚, revealing a minimal SGNH-hydrolase fold. The structure of TAP in complex with a diethyl phosphono moiety (DEP) identified its catalytic triad, Ser10-Asp154-His157, and oxyanion hole, Ser10-Gly44-Asn73. The oxyanion hole of TAP consists of three residues each separated from the other by more than 3.5 A˚, implying that all of them are highly polarized when substrate bound. The catalytic (His)Cϵ1–H⋯O&z.dbnd;C hydrogen bond usually plays a role in the catalytic mechanisms of most serine hydrolases, however, there were none present in SGNH-hydrolases. We propose that the existence of the highly polarized tri-residue-constituted oxyanion hole compensates for the lack of a (His)Cϵ1–H⋯O&z.dbnd;C hydrogen bond. This suggests that members of the SGNH-hydrolase family may employ a unique catalytic mechanism. In addition, most SGNH-hydrolases have low sequence identities and presently there is no clear criterion to define consensus sequence blocks. Through comparison of TAP and the three SGNH-hydrolase structures currently known, we have identified a unique hydrogen bond network which stabilizes the catalytic center: a newly discovered structural feature of SGNH-hydrolases. We have defined these consensus sequence blocks providing a basis for the sub-classification of SGNH-hydrolases. [Copyright &y& Elsevier]

The Escherichia coli thioesterase I specifically catalyzes the deacylation of fatty acyl-CoA thioesters, especially those with long acyl groups (C12-C18). Single crystals of thioesterase I (E.C. 3.1.2.2) from E. coli have been obtained using methoxypolyethylene glycol 5000 (PEG-MME 5K) as a precipitant at room temperature in 21 d. The crystals belong to the tetragonal space group P41212 or its enantiomorph P43212, with unit-cell parameters a = b = 50.85 (7), c 171.5 (1) Å. The crystals diffract to beyond 2.4 Å resolution. There is one molecule of molecular weight 20.5 kDa in the asymmetric unit, with a solvent content of 55%. [ABSTRACT FROM AUTHOR]

Nuclear factor kappaB (NF-kappaB) activation in tumor necrosis factor, interleukin-1, and Toll-like receptor pathways requires Lys63-linked nondegradative polyubiquitination. A20 is a specific feedback inhibitor of NF-kappaB activation in these pathways that possesses dual ubiquitin-editing functions. While the N-terminal domain of A20 is a deubiquitinating enzyme (DUB) for Lys63-linked polyubiquitinated signaling mediators such as TRAF6 and RIP, its C-terminal domain is a ubiquitin ligase (E3) for Lys48-linked degradative polyubiquitination of the same substrates. To elucidate the molecular basis for the DUB activity of A20, we determined its crystal structure and performed a series of biochemical and cell biological studies. The structure reveals the potential catalytic mechanism of A20, which may be significantly different from papain-like cysteine proteases. Ubiquitin can be docked onto a conserved A20 surface; this interaction exhibits charge complementarity and no steric clash. Surprisingly, A20 does not have specificity for Lys63-linked polyubiquitin chains. Instead, it effectively removes Lys63-linked polyubiquitin chains from TRAF6 without dissembling the chains themselves. Our studies suggest that A20 does not act as a general DUB but has the specificity for particular polyubiquitinated substrates to assure its fidelity in regulating NF-kappaB activation in the tumor necrosis factor, interleukin-1, and Toll-like receptor pathways.

In addition to caspase inhibition, X-linked inhibitor of apoptosis (XIAP) induces NF-kappaB and MAP kinase activation during TGF-b and BMP receptor signaling and upon overexpression. Here we show that the BIR1 domain of XIAP, which has no previously ascribed function, directly interacts with TAB1 to induce NF-kappaB activation. TAB1 is an upstream adaptor for the activation of the kinase TAK1, which in turn couples to the NF-kappaB pathway. We report the crystal structures of BIR1, TAB1, and the BIR1/TAB1 complex. The BIR1/TAB1 structure reveals a striking butterfly-shaped dimer and the detailed interaction between BIR1 and TAB1. Structure-based mutagenesis and knockdown of TAB1 show unambiguously that the BIR1/TAB1 interaction is crucial for XIAP-induced TAK1 and NF-kappaB activation. We show that although not interacting with BIR1, Smac, the antagonist for caspase inhibition by XIAP, also inhibits the XIAP/TAB1 interaction. Disruption of BIR1 dimerization abolishes XIAP-mediated NF-kappaB activation, implicating a proximity-induced mechanism for TAK1 activation.

Escherichia coli thioesterase/protease I (TEP-I) belongs to a new subclass of lipolytic enzymes of the serine hydrolase superfamily. Here we report the first direct NMR observation of the formation of the Michaelis complex (MC) between TEP-I and diethyl p-nitrophenyl phosphate (DENP), an active site directed inhibitor of serine protease, and its subsequent conversion to the tetrahedral complex (TC). NMR, ESI-MS, and kinetic data showed that DENP binds to TEP-I in a two-step process, a fast formation of MC followed by a slow conversion to TC. NMR chemical shift perturbation further revealed that perturbations were confined mainly to four conserved segments comprising the active site. Comparable magnitudes of chemical shift perturbations were detected in both steps. The largest chemical shift perturbation occurred around the catalytic Ser(10). In MC, the conformation of the mobile Ser(10) was stabilized, and its amide resonance became observable. From the large chemical shift perturbation upon conversion from MC to TC, we propose that the amide protons of Ser(10) and Gly(44) serve as the oxyanion hole proton donors that stabilize the tetrahedral adduct. The pattern of residues perturbed in both steps suggests a sequential, stepwise structural change upon binding of DENP. The present study also demonstrates the important catalytic roles of conserved residues in the SGNH family of proteins.