Structural and Functional Characterization of the Ureidoacrylate Amidohydrolase RutB from Escherichia coli.

We present a detailed structure-function analysis of the ureidoacrylate amidohydrolase RutB from Eschericha coli, which is an essential enzyme of the Rut pathway for pyrimidine utilization. Crystals of selenomethionine-labeled RutB were produced, which allowed us to determine the first structure of the enzyme at a resolution of 1.9 Å and to identify it as a new member of the isochorismatase-like hydrolase family. RutB was co-crystallized with the substrate analogue ureidopropionate, revealing the mode of substrate binding. Mutation of residues constituting the catalytic triad (D24A, D24N, K133A, C166A, C166S, C166T, C166Y) resulted in complete inactivation of RutB, whereas mutation of other residues close to the active site (Y29F, Y35F, N72A, W74A, W74F, E80A, E80D, S92A, S92T, S92Y, Q105A, Y136A, Y136F) leads to distinct changes of the turnover number (kcat) and/or the Michaelis constant (KM). The results of our structural and mutational studies allowed us to assign specific functions to individual residues and to formulate a plausible reaction mechanism for RutB.

Towards Photochromic Azobenzene-Based Inhibitors for Tryptophan Synthase.

Light regulation of drug molecules has gained growing interest in biochemical and pharmacological research in recent years. In addition, a serious need for novel molecular targets of antibiotics has emerged presently. Herein, the development of a photocontrollable, azobenzene-based antibiotic precursor towards tryptophan synthase (TS), an essential metabolic multienzyme complex in bacteria, is presented. The compound exhibited moderately strong inhibition of TS in its E configuration and five times lower inhibition strength in its Z configuration. A combination of biochemical, crystallographic, and computational analyses was used to characterize the inhibition mode of this compound. Remarkably, binding of the inhibitor to a hitherto-unconsidered cavity results in an unproductive conformation of TS leading to noncompetitive inhibition of tryptophan production. In conclusion, we created a promising lead compound for combatting bacterial diseases, which targets an essential metabolic enzyme, and whose inhibition strength can be controlled with light.

Significance of the Protein Interface Configuration for Allostery in Imidazole Glycerol Phosphate Synthase.

Imidazole glycerol phosphate synthase (ImGPS) from Thermotoga maritima is a model enzyme for studying allostery. The ImGPS complex consists of the cyclase subunit HisF and the glutaminase subunit HisH whose activity is stimulated by substrate binding to HisF in a V-type manner. To investigate the significance of a putative closing hinge motion at the cyclase:glutaminase interface for HisH activity, we replaced residue W123 in HisH with the light-switchable unnatural amino acid phenylalanine-4'-azobenzene (AzoF). Crystal structure analysis employing angle, buried surface area, and distance measurements showed that incorporation of AzoF at this position causes a closing of the interface by ∼18 ± 3%. This slightly different interface configuration results in a much higher catalytic efficiency in unstimulated HisH due to an elevated turnover number. Moreover, the catalytic efficiency of HisH when stimulated by binding of a substrate to HisF was also significantly increased by AzoF incorporation. This was caused by a K-type stimulation that led to a decrease in the apparent dissociation constant for its substrate, glutamine. In addition, AzoF improved the apparent binding of a substrate analogue at the HisF active site. Remarkably, light-induced isomerization of AzoF considerably enhanced these effects. In conclusion, our findings confirm that signal transduction from HisF to HisH in ImGPS involves the closing of the cyclase:glutaminase subunit interface and that incorporation of AzoF at a hinge position reinforces this catalytically relevant conformational change.

Library Selection with a Randomized Repertoire of (ßα)8-Barrel Enzymes Results in Unexpected Induction of Gene Expression.

The potential of the frequently encountered (ßα)8-barrel fold to acquire new functions was tested by an approach combining random mutagenesis and selection in vivo. For this purpose, the genes encoding 52 different phosphate-binding (ßα)8-barrel proteins were subjected to error-prone PCR and cloned into an expression plasmid. The resulting mixed repertoire was used to transform different auxotrophic Escherichia coli strains, each lacking an enzyme with a phosphate-containing substrate. After plating of the different transformants on minimal medium, growth was observed only for two strains, lacking either the gene for the serine phosphatase SerB or the phosphoserine aminotransferase SerC. The same mutants of the E. coli genes nanE (encoding a putative N-acetylmannosamine-6-phosphate 2-epimerase) and pdxJ (encoding the pyridoxine 5'-phosphate synthase) were responsible for rescuing both ΔserB and ΔserC. Unexpectedly, the complementing NanE and PdxJ variants did not catalyze the SerB or SerC reactions in vitro. Instead, RT-qPCR, RNAseq, and transcriptome analysis showed that they rescue the deletions by enlisting the help of endogenous E. coli enzymes HisB and HisC through exclusive up-regulation of histidine operon transcription. While the promiscuous SerB activity of HisB is well-established, our data indicate that HisC is promiscuous for the SerC reaction, as well. The successful rescue of ΔserB and ΔserC through point mutations and recruitment of additional amino acids in NanE and PdxJ provides another example for the adaptability of the (ßα)8-barrel fold.

Structural basis for the enhancement of virulence by viral spindles and their in vivo crystallization.

The great benefits that chemical pesticides have brought to agriculture are partly offset by widespread environmental damage to nontarget species and threats to human health. Microbial bioinsecticides are considered safe and highly specific alternatives but generally lack potency. Spindles produced by insect poxviruses are crystals of the fusolin protein that considerably boost not only the virulence of these viruses but also, in cofeeding experiments, the insecticidal activity of unrelated pathogens. However, the mechanisms by which spindles assemble into ultra-stable crystals and enhance virulence are unknown. Here we describe the structure of viral spindles determined by X-ray microcrystallography from in vivo crystals purified from infected insects. We found that a C-terminal molecular arm of fusolin mediates the assembly of a globular domain, which has the hallmarks of lytic polysaccharide monooxygenases of chitinovorous bacteria. Explaining their unique stability, a 3D network of disulfide bonds between fusolin dimers covalently crosslinks the entire crystalline matrix of spindles. However, upon ingestion by a new host, removal of the molecular arm abolishes this stabilizing network leading to the dissolution of spindles. The released monooxygenase domain is then free to disrupt the chitin-rich peritrophic matrix that protects insects against oral infections. The mode of action revealed here may guide the design of potent spindles as synergetic additives to bioinsecticides.

A comprehensive analysis of the geranylgeranylglyceryl phosphate synthase enzyme family identifies novel members and reveals mechanisms of substrate specificity and quaternary structure organization.

Geranylgeranylglyceryl phosphate synthase (GGGPS) family enzymes catalyse the formation of an ether bond between glycerol-1-phosphate and polyprenyl diphosphates. They are essential for the biosynthesis of archaeal membrane lipids, but also occur in bacterial species, albeit with unknown physiological function. It has been known that there exist two phylogenetic groups (I and II) of GGGPS family enzymes, but a comprehensive study has been missing. We therefore visualized the variability within the family by applying a sequence similarity network, and biochemically characterized 17 representative GGGPS family enzymes regarding their catalytic activities and substrate specificities. Moreover, we present the first crystal structures of group II archaeal and bacterial enzymes. Our analysis revealed that the previously uncharacterized bacterial enzymes from group II have GGGPS activity like the archaeal enzymes and differ from the bacterial group I enzymes that are heptaprenylglyceryl phosphate synthases. The length of the isoprenoid substrate is determined in group II GGGPS enzymes by 'limiter residues' that are different from those in group I enzymes, as shown by site-directed mutagenesis. Most of the group II enzymes form hexamers. We could disrupt these hexamers to stable and catalytically active dimers by mutating a single amino acid that acts as an 'aromatic anchor'.

Ligand structures of synthetic deoxa-pyranosylamines with raucaffricine and strictosidine glucosidases provide structural insights into their binding and inhibitory behaviours.

Insight into the structure and inhibition mechanism of O-ß-d-glucosidases by deoxa-pyranosylamine type inhibitors is provided by X-ray analysis of complexes between raucaffricine and strictosidine glucosidases and N-(cyclohexylmethyl)-, N-(cyclohexyl)- and N-(bromobenzyl)-ß-d-gluco-1,5-deoxa-pyranosylamine. All inhibitors anchored exclusively in the catalytic active site by competition with appropriate enzyme substrates. Thus facilitated prospective elucidation of the binding networks with residues located at <3.9 Å distance will enable the development of potent inhibitors suitable for the production of valuable alkaloid glucosides, raucaffricine and strictosidine, by means of synthesis in Rauvolfia serpentina cell suspension cultures.

TrpB2 enzymes are O-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 ß chain/ß chain-like TrpB1-TrpB2 family (IPR023026). The results showed that TrpB2 enzymes are O-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 O-phospho-l-serine.

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 (ßα)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 (ßα)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.

Crystal structure of the GlnZ-DraG complex reveals a different form of PII-target interaction.

Nitrogen metabolism in bacteria and archaea is regulated by a ubiquitous class of proteins belonging to the P(II)family. P(II) proteins act as sensors of cellular nitrogen, carbon, and energy levels, and they control the activities of a wide range of target proteins by protein-protein interaction. The sensing mechanism relies on conformational changes induced by the binding of small molecules to P(II) and also by P(II) posttranslational modifications. In the diazotrophic bacterium Azospirillum brasilense, high levels of extracellular ammonium inactivate the nitrogenase regulatory enzyme DraG by relocalizing it from the cytoplasm to the cell membrane. Membrane localization of DraG occurs through the formation of a ternary complex in which the P(II) protein GlnZ interacts simultaneously with DraG and the ammonia channel AmtB. Here we describe the crystal structure of the GlnZ-DraG complex at 2.1 Å resolution, and confirm the physiological relevance of the structural data by site-directed mutagenesis. In contrast to other known P(II) complexes, the majority of contacts with the target protein do not involve the T-loop region of P(II). Hence this structure identifies a different mode of P(II) interaction with a target protein and demonstrates the potential for P(II) proteins to interact simultaneously with two different targets. A structural model of the AmtB-GlnZ-DraG ternary complex is presented. The results explain how the intracellular levels of ATP, ADP, and 2-oxoglutarate regulate the interaction between these three proteins and how DraG discriminates GlnZ from its close paralogue GlnB.

Crystal structure of perakine reductase, founding member of a novel aldo-keto reductase (AKR) subfamily that undergoes unique conformational changes during NADPH binding.

Perakine reductase (PR) catalyzes the NADPH-dependent reduction of the aldehyde perakine to yield the alcohol raucaffrinoline in the biosynthetic pathway of ajmaline in Rauvolfia, a key step in indole alkaloid biosynthesis. Sequence alignment shows that PR is the founder of the new AKR13D subfamily and is designated AKR13D1. The x-ray structure of methylated His(6)-PR was solved to 2.31 Å. However, the active site of PR was blocked by the connected parts of the neighbor symmetric molecule in the crystal. To break the interactions and obtain the enzyme-ligand complexes, the A213W mutant was generated. The atomic structure of His(6)-PR-A213W complex with NADPH was determined at 1.77 Å. Overall, PR folds in an unusual α(8)/ß(6) barrel that has not been observed in any other AKR protein to date. NADPH binds in an extended pocket, but the nicotinamide riboside moiety is disordered. Upon NADPH binding, dramatic conformational changes and movements were observed: two additional ß-strands in the C terminus become ordered to form one α-helix, and a movement of up to 24 Å occurs. This conformational change creates a large space that allows the binding of substrates of variable size for PR and enhances the enzyme activity; as a result cooperative kinetics are observed as NADPH is varied. As the founding member of the new AKR13D subfamily, PR also provides a structural template and model of cofactor binding for the AKR13 family.

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 (PdPTE), 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 (Dr0930 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 10(6) 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 PdPTE and the engineered PLL differ significantly in the mode of substrate binding.

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 3 harboring the piperazino[1,2-a]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 3 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.

The atomic structure of baculovirus polyhedra reveals the independent emergence of infectious crystals in DNA and RNA viruses.

Baculoviruses are ubiquitous insect viruses well known for their use as bioinsecticides, gene therapy vectors, and protein expression systems. Overexpression of recombinant proteins in insect cell culture utilizes the strong promoter of the polyhedrin gene. In infected larvae, the polyhedrin protein forms robust intracellular crystals called polyhedra, which protect encased virions for prolonged periods in the environment. Polyhedra are produced by two unrelated families of insect viruses, baculoviruses and cypoviruses. The atomic structure of cypovirus polyhedra revealed an intricate packing of trimers, which are interconnected by a projecting N-terminal helical arm of the polyhedrin molecule. Baculovirus and cypovirus polyhedra share nearly identical lattices, and the N-terminal region of the otherwise unrelated baculovirus polyhedrin protein sequence is also predicted to be alpha-helical. These results suggest homology between the proteins and a common structural basis for viral polyhedra. Here, we present the 2.2-A structure of baculovirus polyhedra determined by x-ray crystallography from microcrystals produced in vivo. We show that the underlying molecular organization is, in fact, very different. Although both polyhedra have nearly identical unit cell dimensions and share I23 symmetry, the polyhedrin molecules are structurally unrelated and pack differently in the crystals. In particular, disulfide bonds and domain-swapped N-terminal domains stabilize the building blocks of baculovirus polyhedra and interlocking C-terminal arms join unit cells together. We show that the N-terminal projecting helical arms have different structural roles in baculovirus and cypovirus polyhedra and conclude that there is no structural evidence for a common evolutionary origin for both classes of polyhedra.

Radiation damage in room-temperature data acquisition with the PILATUS 6M pixel detector.

The first study of room-temperature macromolecular crystallography data acquisition with a silicon pixel detector is presented, where the data are collected in continuous sample rotation mode, with millisecond read-out time and no read-out noise. Several successive datasets were collected sequentially from single test crystals of thaumatin and insulin. The dose rate ranged between ∼ 1320 Gy s(-1) and ∼ 8420 Gy s(-1) with corresponding frame rates between 1.565 Hz and 12.5 Hz. The data were analysed for global radiation damage. A previously unreported negative dose-rate effect is observed in the indicators of global radiation damage, which showed an approximately 75% decrease in D(1/2) at sixfold higher dose rate. The integrated intensity decreases in an exponential manner. Sample heating that could give rise to the enhanced radiation sensitivity at higher dose rate is investigated by collecting data between crystal temperatures of 298 K and 353 K. UV-Vis spectroscopy is used to demonstrate that disulfide radicals and trapped electrons do not accumulate at high dose rates in continuous data collection.

Structure at 1.5 A resolution of cytochrome c(552) with its flexible linker segment, a membrane-anchored protein from Paracoccus denitrificans.

Electron transfer (ET) between the large membrane-integral redox complexes in the terminal part of the respiratory chain is mediated either by a soluble c-type cytochrome, as in mitochondria, or by a membrane-anchored cytochrome c, as described for the ET chain of the bacterium Paracoccus denitrificans. Here, the structure of cytochrome c(552) from P. denitrificans with the linker segment that attaches the globular domain to the membrane anchor is presented. Cytochrome c(552) including the linker segment was crystallized and its structure was determined by molecular replacement. The structural features provide functionally important information. The prediction of the flexibility of the linker region [Berry & Trumpower (1985), J. Biol. Chem. 260, 2458-2467] was confirmed by our crystal structure. The N-terminal region from residues 13 to 31 is characterized by poor electron density, which is compatible with high mobility of this region. This result indicates that this region is highly flexible, which is functionally important for this protein to shuttle electrons between complexes III and IV in the respiratory chain. Zinc present in the crystallization buffer played a key role in the successful crystallization of this protein. It provided rigidity to the long negatively charged flexible loop by coordinating negatively charged residues from two different molecules and by enhancing the crystal contacts.

Mapping the Allosteric Communication Network of Aminodeoxychorismate Synthase.

Allosteric communication between different subunits in metabolic enzyme complexes is of utmost physiological importance but only understood for few systems. We analyzed the structural basis of allostery in aminodeoxychorismate synthase (ADCS), which is a member of the family of glutamine amidotransferases and catalyzes the committed step of the folate biosynthetic pathway. ADCS consists of the synthase subunit PabB and the glutaminase subunit PabA, which is allosterically stimulated by the presence of the PabB substrate chorismate. We first solved the crystal structure of a PabA subunit at 1.9-Å resolution. Based on this structure and the known structure of PabB, we computed an atomic model for the ADCS complex. We then used alanine scanning to test the functional role of 59 conserved residues located between the active sites of PabB and PabA. Steady-state kinetic characterization revealed four branches of a conserved network of mainly charged residues that propagate the signal from chorismate at the PabB active site to the PabA active site. The branches eventually lead to activity-inducing transformations at (i) the oxyanion hole motif, (ii) the catalytic Cys-His-Glu triad, and (iii) glutamine binding residues at the PabA active site. We compare our findings with previously postulated activation mechanisms of different glutamine amidotransferases and propose a unifying regulation mechanism for this ubiquitous family of enzymes.

Light Regulation of Enzyme Allostery through Photo-responsive Unnatural Amino Acids.

Imidazole glycerol phosphate synthase (ImGPS) is an allosteric bienzyme complex in which substrate binding to the synthase subunit HisF stimulates the glutaminase subunit HisH. To control this stimulation with light, we have incorporated the photo-responsive unnatural amino acids phenylalanine-4'-azobenzene (AzoF), o-nitropiperonyl-O-tyrosine (NPY), and methyl-o-nitropiperonyllysine (mNPK) at strategic positions of HisF. The light-mediated isomerization of AzoF at position 55 (fS55AzoFE ↔ fS55AzoFZ) resulted in a reversible 10-fold regulation of HisH activity. The light-mediated decaging of NPY at position 39 (fY39NPY → fY39) and of mNPK at position 99 (fK99mNPK → fK99) led to a 4- to 6-fold increase of HisH activity. Molecular dynamics simulations explained how the unnatural amino acids interfere with the allosteric machinery of ImGPS and revealed additional aspects of HisH stimulation in wild-type ImGPS. Our findings show that unnatural amino acids can be used as a powerful tool for the spatiotemporal control of a central metabolic enzyme complex by light.

Structure at 1.3 A resolution of Rhodothermus marinus caa(3) cytochrome c domain.

The cytochrome c domain of subunit II from the Rhodothermus marinus caa(3) HiPIP:oxygen oxidoreductase, a member of the superfamily of heme-copper-containing terminal oxidases, was produced in Escherichia coli and characterised. The recombinant protein, which shows the same optical absorption and redox properties as the corresponding domain in the holo enzyme, was crystallized and its structure was determined to a resolution of 1.3 A by the multiwavelength anomalous dispersion (MAD) technique using the anomalous dispersion of the heme iron atom. The model was refined to final R(cryst) and R(free) values of 13.9% and 16.7%, respectively. The structure reveals the insertion of two short antiparallel beta-strands forming a small beta-sheet, an interesting variation of the classical all alpha-helical cytochrome c fold. This modification appears to be common to all known caa(3)-type terminal oxidases, as judged by comparative modelling and by analyses of the available amino acid sequences for these enzymes. This is the first high-resolution crystal structure reported for a cytochrome c domain of a caa(3)-type terminal oxidase. The R.marinus caa(3) uses HiPIP as the redox partner. The calculation of the electrostatic potential at the molecular surface of this extra C-terminal domain provides insights into the binding to its redox partner on one side and its interaction with the remaining subunit II on the other side.

Ancestral Tryptophan Synthase Reveals Functional Sophistication of Primordial Enzyme Complexes.

Modern enzyme complexes are characterized by a high catalytic efficiency and allosteric communication between the constituting protein subunits. We were interested in whether primordial enzyme complexes from extinct species displayed a similar degree of functional sophistication. To this end, we used ancestral sequence reconstruction to resurrect the α and ß subunits of the tryptophan synthase (TS) complex from the last bacterial common ancestor (LBCA), which presumably existed more than 3.4 billion years ago. We show that the LBCA TS subunits are thermostable and exhibit high catalytic activity. Moreover, they form a complex with αßßα stoichiometry whose crystal structure is similar to that of modern TS. Kinetic analysis revealed that the reaction intermediate indole is channeled from the α to the ß subunits and suggests that allosteric communication already occurred in LBCA TS.