TrpB2 Enzymes are <i>O</i>‑Phospho‑l‑serine Dependent Tryptophan Synthases

The rapid increase of the number of sequenced genomes asks for the functional annotation of the encoded enzymes. We used a combined computational–structural approach to determine the function of the TrpB2 subgroup of the tryptophan synthase β chain/β chain-like TrpB1–TrpB2 family (IPR023026). The results showed that TrpB2 enzymes are <i>O</i>-phospho-l-serine dependent tryptophan synthases, whereas TrpB1 enzymes catalyze the l-serine dependent synthesis of tryptophan. We found a single residue being responsible for the different substrate specificities of TrpB1 and TrpB2 and confirmed this finding by mutagenesis studies and crystallographic analysis of a TrpB2 enzyme with bound <i>O</i>-phospho-l-serine

Structures of Alkaloid Biosynthetic Glucosidases Decode Substrate Specificity

Two similar enzymes with different biosynthetic function in one species have evolved to catalyze two distinct reactions. X-ray structures of both enzymes help reveal their most important differences. The <i>Rauvolfia</i> alkaloid biosynthetic network harbors two <i>O</i>-glucosidases: raucaffricine glucosidase (RG), which hydrolyses raucaffricine to an intermediate downstream in the ajmaline pathway, and strictosidine glucosidase (SG), which operates upstream. RG converts strictosidine, the substrate of SG, but SG does not accept raucaffricine. Now elucidation of crystal structures of RG, inactive RG-E186Q mutant, and its complexes with ligands dihydro-raucaffricine and secologanin reveals that it is the “wider gate” of RG that allows strictosidine to enter the catalytic site, whereas the “slot-like” entrance of SG prohibits access by raucaffricine. Trp392 in RG and Trp388 in SG control the gate shape and acceptance of substrates. Ser390 directs the conformation of Trp392. 3D structures, supported by site-directed mutations and kinetic data of RG and SG, provide a structural and catalytic explanation of substrate specificity and deeper insights into <i>O</i>-glucosidase chemistry

Structural Analysis of the Binding of Type I, I_1/2, and II Inhibitors to Eph Tyrosine Kinases

We have solved the crystal structures of the EphA3 tyrosine kinase in complex with nine small-molecule inhibitors, which represent five different chemotypes and three main binding modes, i.e., types I and I_1/2 (DFG in) and type II (DFG out). The three structures with type I_1/2 inhibitors show that the higher affinity with respect to type I is due to an additional polar group (hydroxyl or pyrazole ring of indazole) which is fully buried and is involved in the same hydrogen bonds as the (urea or amide) linker of the type II inhibitors. Overall, the type I and type II binding modes belong to the lock-and-key and induced fit mechanism, respectively. In the type II binding, the scaffold in contact with the hinge region influences the position of the Phe765 side chain of the DFG motif and the orientation of the Gly-rich loop. The binding mode of Birb796 in the EphA3 kinase does not involve any hydrogen bond with the hinge region, which is different from the Birb796/p38 MAP kinase complex. Our structural analysis emphasizes the importance of accounting for structural plasticity of the ATP binding site in the design of type II inhibitors of tyrosine kinases

Scaffold Tailoring by a Newly Detected Pictet–Spenglerase Activity of Strictosidine Synthase: From the Common Tryptoline Skeleton to the Rare Piperazino-indole Framework

The Pictet–Spenglerase strictosidine synthase (STR1) has been recognized as a key enzyme in the biosynthesis of some 2000 indole alkaloids in plants, some with high therapeutic value. In this study, a novel function of STR1 has been detected which allows for the first time a simple enzymatic synthesis of the strictosidine analogue <b>3</b> harboring the piperazino­[1,2-<i>a</i>]­indole (PI) scaffold and to switch from the common tryptoline (hydrogenated carboline) to the rare PI skeleton. Insight into the reaction is provided by X-ray crystal analysis and modeling of STR1 ligand complexes. STR1 presently provides exclusively access to <b>3</b> and can act as a source to generate by chemoenzymatic approaches libraries of this novel class of alkaloids which may have new biological activities. Synthetic or natural monoterpenoid alkaloids with the PI core have not been reported before

Evidence for the Existence of Elaborate Enzyme Complexes in the Paleoarchean Era

Due to the lack of macromolecular fossils, the enzymatic repertoire of extinct species has remained largely unknown to date. In an attempt to solve this problem, we have characterized a cyclase subunit (HisF) of the imidazole glycerol phosphate synthase (ImGP-S), which was reconstructed from the era of the last universal common ancestor of cellular organisms (LUCA). As observed for contemporary HisF proteins, the crystal structure of LUCA-HisF adopts the (βα)₈-barrel architecture, one of the most ancient folds. Moreover, LUCA-HisF (i) resembles extant HisF proteins with regard to internal 2-fold symmetry, active site residues, and a stabilizing salt bridge cluster, (ii) is thermostable and shows a folding mechanism similar to that of contemporary (βα)₈-barrel enzymes, (iii) displays high catalytic activity, and (iv) forms a stable and functional complex with the glutaminase subunit (HisH) of an extant ImGP-S. Furthermore, we show that LUCA-HisF binds to a reconstructed LUCA-HisH protein with high affinity. Our findings suggest that the evolution of highly efficient enzymes and enzyme complexes has already been completed in the LUCA era, which means that sophisticated catalytic concepts such as substrate channeling and allosteric communication existed already 3.5 billion years ago

Molecular Engineering of Organophosphate Hydrolysis Activity from a Weak Promiscuous Lactonase Template

Rapid evolution of enzymes provides unique molecular insights into the remarkable adaptability of proteins and helps to elucidate the relationship between amino acid sequence, structure, and function. We interrogated the evolution of the phospho­triesterase from Pseudomonas diminuta (<i>Pd</i>PTE), which hydrolyzes synthetic organophosphates with remarkable catalytic efficiency. PTE is thought to be an evolutionarily “young” enzyme, and it has been postulated that it has evolved from members of the phospho­triesterase-like lactonase (PLL) family that show promiscuous organophosphate-degrading activity. Starting from a weakly promiscuous PLL scaffold (<i>Dr</i>0930 from Deinococcus radiodurans), we designed an extremely efficient organophosphate hydrolase (OPH) with broad substrate specificity using rational and random mutagenesis in combination with in vitro activity screening. The OPH activity for seven organophosphate substrates was simultaneously enhanced by up to 5 orders of magnitude, achieving absolute values of catalytic efficiencies up to 10⁶ M^–1 s^–1. Structural and computational analyses identified the molecular basis for the enhanced OPH activity of the engineered PLL variants and demonstrated that OPH catalysis in <i>Pd</i>PTE and the engineered PLL differ significantly in the mode of substrate binding