Altering Active-Site Loop Dynamics Enhances Standalone Activity of the Tryptophan Synthase Alpha Subunit.

The α-subunit (TrpA) of the allosterically regulated bifunctional tryptophan synthase αββα enzyme catalyzes the retro-aldol cleavage of indole-glycerol phosphate (IGP) to d-glyceraldehyde 3-phosphate (G3P) and indole. The activity of the enzyme is highly dependent on the β-subunit (TrpB), which allosterically regulates and activates TrpA for enhanced function. This contrasts with the homologous BX1 enzyme from that can catalyze the same reaction as TrpA without requiring the presence of any additional binding partner.

Conformational Modulation of a Mobile Loop Controls Catalysis in the (βα)-Barrel Enzyme of Histidine Biosynthesis HisF.

The overall significance of loop motions for enzymatic activity is generally accepted. However, it has largely remained unclear whether and how such motions can control different steps of catalysis. We have studied this problem on the example of the mobile active site βα-loop (loop1) of the (βα)-barrel enzyme HisF, which is the cyclase subunit of imidazole glycerol phosphate synthase.

Validation of aminodeoxychorismate synthase and anthranilate synthase as novel targets for bispecific antibiotics inhibiting conserved protein-protein interactions.

Unlabelled: Multi-resistant bacteria are a rapidly emerging threat to modern medicine. It is thus essential to identify and validate novel antibacterial targets that promise high robustness against resistance-mediating mutations. This can be achieved by simultaneously targeting several conserved function-determining protein-protein interactions in enzyme complexes from prokaryotic primary metabolism.

Improving enzyme functional annotation by integrating in vitro and in silico approaches: The example of histidinol phosphate phosphatases.

Advances in sequencing technologies have led to a rapid growth of public protein sequence databases, whereby the fraction of proteins with experimentally verified function continuously decreases. This problem is currently addressed by automated functional annotations with computational tools, which however lack the accuracy of experimental approaches and are susceptible to error propagation. Here, we present an approach that combines the efficiency of functional annotation by in silico methods with the rigor of enzyme characterization in vitro.

Structural and Functional Characterization of the Ureidoacrylate Amidohydrolase RutB from .

We present a detailed structure-function analysis of the ureidoacrylate amidohydrolase RutB from , 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.

Experimental and computational analysis of the ancestry of an evolutionary young enzyme from histidine biosynthesis.

The conservation of fold and chemistry of the enzymes associated with histidine biosynthesis suggests that this pathway evolved prior to the diversification of Bacteria, Archaea, and Eukaryotes. The only exception is the histidinol phosphate phosphatase (HolPase). So far, non-homologous HolPases that possess distinct folds and belong to three different protein superfamilies have been identified in various phylogenetic clades.

Photoswitching of Feedback Inhibition by Tryptophan in Anthranilate Synthase.

The artificial regulation of enzymatic activity by light is an important goal of synthetic biology that can be achieved by the incorporation of light-responsive noncanonical amino acids via genetic code expansion. Here, we apply this concept to anthranilate synthase from (stTrpE). This enzyme catalyzes the first step of tryptophan biosynthesis, and its activity is feedback-inhibited by the binding of the end-product of the pathway to an allosteric site.

Identification and Experimental Validation of Distal Activity-Enhancing Mutations in Tryptophan Synthase.

Allostery is a central mechanism for the regulation of multi-enzyme complexes. The mechanistic basis that drives allosteric regulation is poorly understood but harbors key information for enzyme engineering. In the present study, we focus on the tryptophan synthase complex that is composed of TrpA and TrpB subunits, which allosterically activate each other.

Molecular basis for the allosteric activation mechanism of the heterodimeric imidazole glycerol phosphate synthase complex.

Imidazole glycerol phosphate synthase (HisFH) is a heterodimeric bienzyme complex operating at a central branch point of metabolism. HisFH is responsible for the HisH-catalyzed hydrolysis of glutamine to glutamate and ammonia, which is then used for a cyclase reaction by HisF. The HisFH complex is allosterically regulated but the underlying mechanism is not well understood.

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.

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

Imidazole glycerol phosphate synthase (ImGPS) from 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).

Quaternary Structure of the Tryptophan Synthase α-Subunit Homolog BX1 from .

BX1 from (zmBX1) is an enzyme of plant secondary metabolism that generates indole for the synthesis of plant defensins. It is a homologue of the tryptophan synthase α-subunit, TrpA. Whereas TrpA itself is a monomer in solution, zmBX1 is dimeric, confirmed in our work by native MS.

Analysis of allosteric communication in a multienzyme complex by ancestral sequence reconstruction.

Tryptophan synthase (TS) is a heterotetrameric αββα complex. It is characterized by the channeling of the reaction intermediate indole and the mutual activation of the α-subunit TrpA and the β-subunit TrpB via a complex allosteric network. We have analyzed this allosteric network by means of ancestral sequence reconstruction (ASR), which is an in silico method to resurrect extinct ancestors of modern proteins.

Light-Regulation of Tryptophan Synthase by Combining Protein Design and Enzymology.

The spatiotemporal control of enzymes by light is of growing importance for industrial biocatalysis. Within this context, the photo-control of allosteric interactions in enzyme complexes, common to practically all metabolic pathways, is particularly relevant. A prominent example of a metabolic complex with a high application potential is tryptophan synthase from (TS), in which the constituting TrpA and TrpB subunits mutually stimulate each other via a sophisticated allosteric network.

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

The potential of the frequently encountered (βα)-barrel fold to acquire new functions was tested by an approach combining random mutagenesis and selection . For this purpose, the genes encoding 52 different phosphate-binding (βα)-barrel proteins were subjected to error-prone PCR and cloned into an expression plasmid. The resulting mixed repertoire was used to transform different auxotrophic strains, each lacking an enzyme with a phosphate-containing substrate.

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 (fS55AzoF ↔ fS55AzoF) resulted in a reversible 10-fold regulation of HisH activity.

Prediction of quaternary structure by analysis of hot spot residues in protein-protein interfaces: the case of anthranilate phosphoribosyltransferases.

It is an important goal of computational biology to correctly predict the association state of a protein based on its amino acid sequence and the structures of known homologues. We have pursued this goal on the example of anthranilate phosphoribosyltransferase (AnPRT), an enzyme that is involved in the biosynthesis of the amino acid tryptophan. Firstly, known crystal structures of naturally occurring homodimeric AnPRTs were analyzed using the Protein Interfaces, Surfaces, and Assemblies (PISA) service of the European Bioinformatics Institute (EBI).

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.

A Fold-Independent Interface Residue Is Crucial for Complex Formation and Allosteric Signaling in Class I Glutamine Amidotransferases.

The members of the glutamine amidotransferase (GATase) family catalyze the incorporation of ammonia within numerous metabolic pathways and can be categorized in two classes. Here, we concentrated on class I GATases, which are heteromeric enzyme complexes consisting of synthase subunits and glutaminase subunits with a catalytic Cys-His-Glu triad. Glutamine hydrolysis at the glutaminase subunit is (i) dependent on the formation of tight synthase-glutaminase complexes and (ii) allosterically coupled to the presence of the substrate at the synthase subunit.

Generation of a Stand-Alone Tryptophan Synthase α-Subunit by Mimicking an Evolutionary Blueprint.

The αββα tryptophan synthase (TS), which is part of primary metabolism, is a paradigm for allosteric communication in multienzyme complexes. In particular, the intrinsically low catalytic activity of the α-subunit TrpA is stimulated several hundredfold through the interaction with the β-subunit TrpB1. The BX1 protein from Zea mays (zmBX1), which is part of secondary metabolism, catalyzes the same reaction as that of its homologue TrpA, but with high activity in the absence of an interaction partner.

Functional characterisation of two Δ12-desaturases demonstrates targeted production of linoleic acid as pheromone precursor in .

Insect pheromones are often derived from fatty acid metabolism. Fatty acid desaturases, enzymes introducing double bonds into fatty acids, are crucial for the biosynthesis of these chemical signals. Δ12-desaturases catalyse the biosynthesis of linoleic acid by introducing a second double bond into oleic acid, but have been identified in only a few animal species.

Mapping key amino acid residues for the epimerase efficiency and stereospecificity of the sex pheromone biosynthetic short-chain dehydrogenases/reductases of Nasonia.

Males of the parasitic wasp genus Nasonia use blends of chiral hydroxylactones as sex pheromones to attract conspecific females. Whereas all Nasonia species use a mixture of (4R,5S)-5-hydroxy-4-decanolide (RS) and 4-methylquinazoline (MQ) as sex pheromones, Nasonia vitripennis evolved (4R,5R)-5-hydroxy-4-decanolide (RR) as an extra sex pheromone component. We recently identified and functionally characterized three short-chain dehydrogenases/reductases (SDRs) NV10127, NV10128, and NV10129 that are capable of catalyzing the epimerization of RS to RR via (4R)-5-oxo-4-decanolide (ODL) as intermediate.