X-ray structures of checkpoint kinase 2 in complex with inhibitors that target its gatekeeper-dependent hydrophobic pocket

AbstractThe serine/threonine checkpoint kinase 2 (Chk2) is an attractive molecular target for the development of small molecule inhibitors to treat cancer. Here, we report the rational design of Chk2 inhibitors that target the gatekeeper-dependent hydrophobic pocket located behind the adenine-binding region of the ATP-binding site. These compounds exhibit IC50 values in the low nanomolar range and are highly selective for Chk2 over Chk1. X-ray crystallography was used to determine the structures of the inhibitors in complex with the catalytic kinase domain of Chk2 to verify their modes of binding

The Convergence of the Hedgehog/Intein Fold in Different Protein Splicing Mechanisms

Protein splicing catalyzed by inteins utilizes many different combinations of amino-acid types at active sites. Inteins have been classified into three classes based on their characteristic sequences. We investigated the structural basis of the protein splicing mechanism of class 3 inteins by determining crystal structures of variants of a class 3 intein from Mycobacterium chimaera and molecular dynamics simulations, which suggested that the class 3 intein utilizes a different splicing mechanism from that of class 1 and 2 inteins. The class 3 intein uses a bond cleavage strategy reminiscent of proteases but share the same Hedgehog/INTein (HINT) fold of other intein classes. Engineering of class 3 inteins from a class 1 intein indicated that a class 3 intein would unlikely evolve directly from a class 1 or 2 intein. The HINT fold appears as structural and functional solution for trans-peptidyl and trans-esterification reactions commonly exploited by diverse mechanisms using different combinations of amino-acid types for the active-site residues

Crystallographic, spectroscopic, and computational analysis of a flavin C4a-oxygen adduct in choline oxidase

Flavin C4a-OO(H) and C4a-OH adducts are critical intermediates proposed in many flavoenzyme reaction mechanisms, but they are rarely detected even by rapid transient kinetics methods. We observe a trapped flavin C4a-OH or C4a-OO(H) adduct by single-crystal spectroscopic methods and in the 1.86 A resolution X-ray crystal structure of choline oxidase. The microspectrophotometry results show that the adduct forms rapidly in situ at 100 K upon exposure to X-rays. Density functional theory calculations establish the electronic structures for the flavin C4a-OH and C4a-OO(H) adducts and estimate the stabilization energy of several active site hydrogen bonds deduced from the crystal structure. We propose that the enzyme-bound FAD is reduced in the X-ray beam. The aerobic crystals then form either a C4a-OH or C4a-OO(H) adduct, but an insufficient proton inventory prevents their decay at cryogenic temperatures

Characterization of a broadly specific cadaverine N-hydroxylase involved in desferrioxamine B biosynthesis in Streptomyces sviceus.

N-hydroxylating flavin-dependent monooxygenases (FMOs) are involved in the biosynthesis of hydroxamate siderophores, playing a key role in microbial virulence. Herein, we report the first structural and kinetic characterization of a novel alkyl diamine N-hydroxylase DesB from Streptomyces sviceus (SsDesB). This enzyme catalyzes the first committed step in the biosynthesis of desferrioxamine B, a clinical drug used to treat iron overload disorders. X-ray crystal structures of the SsDesB holoenzyme with FAD and the ternary complex with bound NADP+ were solved at 2.86 Å and 2.37 Å resolution, respectively, providing a structural view of the active site environment. SsDesB crystallized as a tetramer and the structure of the individual protomers closely resembles the structures of homologous N-hydroxylating FMOs from Erwinia amylovora (DfoA), Pseudomonas aeruginosa (PvdA), and Aspergillus fumigatus (SidA). Using NADPH oxidation, oxygen consumption, and product formation assays, kinetic parameters were determined for various substrates with SsDesB. SsDesB exhibited typical saturation kinetics with substrate inhibition at high concentrations of NAD(P)H as well as cadaverine. The apparent kcat values for NADPH in steady-state NADPH oxidation and oxygen consumption assays were 0.28 ± 0.01 s-1 and 0.24 ± 0.01 s-1, respectively. However, in product formation assays used to measure the rate of N-hydroxylation, the apparent kcat for NADPH (0.034 ± 0.008 s-1) was almost 10-fold lower under saturating FAD and cadaverine concentrations, reflecting an uncoupled reaction, and the apparent NADPH KM was 33 ± 24 μM. Under saturating FAD and NADPH concentrations, the apparent kcat and KM for cadaverine in Csaky assays were 0.048 ± 0.004 s-1 and 19 ± 9 μM, respectively. SsDesB also N-hydroxylated putrescine, spermidine, and L-lysine substrates but not alkyl (di)amines that were branched or had fewer than four methylene units in an alkyl chain. These data demonstrate that SsDesB has wider substrate scope compared to other well-studied ornithine and lysine N-hydroxylases, making it an amenable biocatalyst for the production of desferrioxamine B, derivatives, and other N-substituted products