Preparation, assay, and application of 4-fluorothreonine transaldolase from Streptomyces sp. MA37 for ß-hydroxyl amino acid derivatives.

ß-Hydroxy-α-amino acids (ßHAAs) are an essential class of building blocks of therapeutically important compounds and complex natural products. They contain two chiral centers at Cα and Cß positions, resulting in four possible diastereoisomers. Many innovative asymmetric syntheses have been developed to access structurally diverse ßHAAs. The main challenge, however, is the control of the relative and absolute stereochemistry of the asymmetric carbons in a sustainable way. In this respect, there has been considerable attention focused on the chemoenzymatic synthesis of ßHAAs via a one-step process. Nature has evolved different enzymatic routes to produce these valuable ßHAAs. Among these naturally occurring transformations, L-threonine transaldolases present potential biocatalysts to generate ßHAAs in situ. 4-Fluorothreonine transaldolase from Streptomyces sp. MA37 (FTaseMA) catalyzes the cross-over transaldolation reaction between L-Thr and fluoroacetaldehyde to give 4-fluorothreonine and acetaldehyde (Ad). It has been demonstrated that FTaseMA displays considerable substrate plasticity toward structurally diverse aldehyde acceptors, leading to the production of various ßHAAs. In this chapter, we describe methods for the preparation of FTaseMA, and the chemoenzymatic synthesis of ßHAAs from various aldehydes and L-Thr using FTaseMA.

In Solution Identification of the Lysine-Cysteine Redox Switch with a NOS Bridge in Transaldolase by Sulfur K-Edge X-ray Absorption Spectroscopy.

A novel covalent post-translational modification (lysine-NOS-cysteine) was discovered in proteins, initially in the enzyme transaldolase of Neisseria gonorrhoeae (NgTAL) [Nature 2021, 593, 460-464], acting as a redox switch. The identification of this novel linkage in solution was unprecedented until now. We present detection of the NOS redox switch in solution using sulfur K-edge X-ray absorption spectroscopy (XAS). The oxidized NgTAL spectrum shows a distinct shoulder on the low-energy side of the rising edge, corresponding to a dipole-allowed transition from the sulfur 1s core to the unoccupied σ* orbital of the S-O group in the NOS bridge. This feature is absent in the XAS spectrum of reduced NgTAL, where Lys-NOS-Cys is absent. Our experimental and calculated XAS data support the presence of a NOS bridge in solution, thus potentially facilitating future studies on enzyme activity regulation mediated by the NOS redox switches, drug discovery, biocatalytic applications, and protein design.

Structure and mechanism of sulfofructose transaldolase, a key enzyme in sulfoquinovose metabolism.

Sulfoquinovose (SQ) is a key component of plant sulfolipids (sulfoquinovosyl diacylglycerols) and a major environmental reservoir of biological sulfur. Breakdown of SQ is achieved by bacteria through the pathways of sulfoglycolysis. The sulfoglycolytic sulfofructose transaldolase (sulfo-SFT) pathway is used by gut-resident firmicutes and soil saprophytes. After isomerization of SQ to sulfofructose (SF), the namesake enzyme catalyzes the transaldol reaction of SF transferring dihydroxyacetone to 3C/4C acceptors to give sulfolactaldehyde and fructose-6-phosphate or sedoheptulose-7-phosphate. We report the 3D cryo-EM structure of SF transaldolase from Bacillus megaterium in apo and ligand bound forms, revealing a decameric structure formed from two pentameric rings of the protomer. We demonstrate a covalent "Schiff base" intermediate formed by reaction of SF with Lys89 within a conserved Asp-Lys-Glu catalytic triad and defined by an Arg-Trp-Arg sulfonate recognition triad. The structural characterization of the signature enzyme of the sulfo-SFT pathway provides key insights into molecular recognition of the sulfonate group of sulfosugars.

A lysine-cysteine redox switch with an NOS bridge regulates enzyme function.

Disulfide bonds between cysteine residues are important post-translational modifications in proteins that have critical roles for protein structure and stability, as redox-active catalytic groups in enzymes or allosteric redox switches that govern protein function1-4. In addition to forming disulfide bridges, cysteine residues are susceptible to oxidation by reactive oxygen species, and are thus central not only to the scavenging of these but also to cellular signalling and communication in biological as well as pathological contexts5,6. Oxidized cysteine species are highly reactive and may form covalent conjugates with, for example, tyrosines in the active sites of some redox enzymes7,8. However, to our knowledge, regulatory switches with covalent crosslinks other than disulfides have not previously been demonstrated. Here we report the discovery of a covalent crosslink between a cysteine and a lysine residue with a NOS bridge that serves as an allosteric redox switch in the transaldolase enzyme of Neisseria gonorrhoeae, the pathogen that causes gonorrhoea. X-ray structure analysis of the protein in the oxidized and reduced state reveals a loaded-spring mechanism that involves a structural relaxation upon redox activation, which is propagated from the allosteric redox switch at the protein surface to the active site in the protein interior. This relaxation leads to a reconfiguration of key catalytic residues and elicits an increase in enzymatic activity of several orders of magnitude. The redox switch is highly conserved in related transaldolases from other members of the Neisseriaceae; for example, it is present in the transaldolase of Neisseria meningitides (a pathogen that is the primary cause of meningitis and septicaemia in children). We surveyed the Protein Data Bank and found that the NOS bridge exists in diverse protein families across all domains of life (including Homo sapiens) and that it is often located at catalytic or regulatory hotspots. Our findings will inform strategies for the design of proteins and peptides, as well as the development of new classes of drugs and antibodies that target the lysine-cysteine redox switch9,10.

Transaldolase in Bacillus methanolicus: biochemical characterization and biological role in ribulose monophosphate cycle.

BACKGROUND: The Gram-positive facultative methylotrophic bacterium Bacillus methanolicus uses the sedoheptulose-1,7-bisphosphatase (SBPase) variant of the ribulose monophosphate (RuMP) cycle for growth on the C1 carbon source methanol. Previous genome sequencing of the physiologically different B. methanolicus wild-type strains MGA3 and PB1 has unraveled all putative RuMP cycle genes and later, several of the RuMP cycle enzymes of MGA3 have been biochemically characterized. In this study, the focus was on the characterization of the transaldolase (Ta) and its possible role in the RuMP cycle in B. methanolicus. RESULTS: The Ta genes of B. methanolicus MGA3 and PB1 were recombinantly expressed in Escherichia coli, and the gene products were purified and characterized. The PB1 Ta protein was found to be active as a homodimer with a molecular weight of 54 kDa and displayed KM of 0.74 mM and Vmax of 16.3 U/mg using Fructose-6 phosphate as the substrate. In contrast, the MGA3 Ta gene, which encodes a truncated Ta protein lacking 80 amino acids at the N-terminus, showed no Ta activity. Seven different mutant genes expressing various full-length MGA3 Ta proteins were constructed and all gene products displayed Ta activities. Moreover, MGA3 cells displayed Ta activities similar as PB1 cells in crude extracts. CONCLUSIONS: While it is well established that B. methanolicus can use the SBPase variant of the RuMP cycle this study indicates that B. methanolicus possesses Ta activity and may also operate the Ta variant of the RuMP.

Novel mode of inhibition by D-tagatose 6-phosphate through a Heyns rearrangement in the active site of transaldolase B variants.

Transaldolase B (TalB) and D-fructose-6-phosphate aldolase A (FSAA) from Escherichia coli are C-C bond-forming enzymes. Using kinetic inhibition studies and mass spectrometry, it is shown that enzyme variants of FSAA and TalB that exhibit D-fructose-6-phosphate aldolase activity are inhibited covalently and irreversibly by D-tagatose 6-phosphate (D-T6P), whereas no inhibition was observed for wild-type transaldolase B from E. coli. The crystal structure of the variant TalB(F178Y) with bound sugar phosphate was solved to a resolution of 1.46 Å and revealed a novel mode of covalent inhibition. The sugar is bound covalently via its C2 atom to the ℇ-NH2 group of the active-site residue Lys132. It is neither bound in the open-chain form nor as the closed-ring form of D-T6P, but has been converted to ß-D-galactofuranose 6-phosphate (D-G6P), a five-membered ring structure. The furanose ring of the covalent adduct is formed via a Heyns rearrangement and subsequent hemiacetal formation. This reaction is facilitated by Tyr178, which is proposed to act as acid-base catalyst. The crystal structure of the inhibitor complex is compared with the structure of the Schiff-base intermediate of TalB(E96Q) formed with the substrate D-fructose 6-phosphate determined to a resolution of 2.20 Å. This comparison highlights the differences in stereochemistry at the C4 atom of the ligand as an essential determinant for the formation of the inhibitor adduct in the active site of the enzyme.

Converting Transaldolase into Aldolase through Swapping of the Multifunctional Acid-Base Catalyst: Common and Divergent Catalytic Principles in F6P Aldolase and Transaldolase.

Transaldolase (TAL) and fructose-6-phosphate aldolase (FSA) both belong to the class I aldolase family and share a high degree of structural similarity and sequence identity. The molecular basis of the different reaction specificities (transferase vs aldolase) has remained enigmatic. A notable difference between the active sites is the presence of either a TAL-specific Glu (Gln in FSA) or a FSA-specific Tyr (Phe in TAL). Both residues seem to have analoguous multifunctional catalytic roles but are positioned at different faces of the substrate locale. We have engineered a TAL double variant (Glu to Gln and Phe to Tyr) with an active site resembling that of FSA. This variant indeed exhibits aldolase activity as its main activity with a catalytic efficiency even larger than that of authentic FSA, while TAL activity is greatly impaired. Structural analysis of this variant in complex with the dihydroxyacetone Schiff base formed upon substrate cleavage identifies the introduced Tyr (genuine in FSA) to catalyze protonation of the central carbanion-enamine intermediate as a key determinant of the aldolase reaction. Our studies pinpoint that the Glu in TAL and the Tyr in FSA, although located at different positions at the active site, similarly act as bona fide acid-base catalysts in numerous catalytic steps, including substrate binding, dehydration of the carbinolamine, and substrate cleavage. We propose that the different spatial positions of the multifunctional Glu in TAL and of the corresponding multifunctional Tyr in FSA relative to the substrate locale are critically controlling reaction specificity through either unfavorable (TAL) or favorable (FSA) geometry of proton transfer onto the common carbanion-enamine intermediate. The presence of both potential acid-base residues, Glu and Tyr, in the active site of TAL has deleterious effects on substrate binding and cleavage, most likely resulting from a differently organized H-bonding network. Large-scale motions of the protein associated with opening and closing of the active site that seem to bear relevance for catalysis are observed as covalent intermediates are exclusively observed in the "closed" conformation of the active site. Pre-steady-state kinetics are used to monitor catalytic processes and structural transitions and to refine the kinetic framework of TAL catalysis.

Two transaldolase isogenes from Moniliella megachiliensis behave in a different way depending on the stress class.

We cloned and sequenced two transaldolase genes from Moniliella megachiliensis, a microorganism known to produce a significant amount of erythritol under hyper-osmotic stress. The amino acid sequences encoded by these two genes (MmTAL1, MmTAL2) showed 72% homology to each other. An AP-1 (ap response element) associated with oxidative stress was found in the promoter region of MmTAL1, while four STREs (stress response element) associated with osmotic stress were found in the promoter region of MmTAL2. In early-stage cultivation (up to 2 h), MmTAL1 was specifically expressed in response to oxidative stress generated by the presence of 0.15 mM menadione; expression level 3-fold higher than before stress loading. MmTAL2 was expressed in response to osmotic stress caused by 1.2 M NaCl; expression level was 21-fold higher than stress-free control. Erythritol accumulated intracellularly under osmotic and oxidative stress, approximately 30-fold and 35-fold, respectively. We therefore concluded that M. megachiliensis selectively uses two isogenes and produces erythritol during early-stage response to stress, depending on the type of environmental stress.

Sweet siblings with different faces: the mechanisms of FBP and F6P aldolase, transaldolase, transketolase and phosphoketolase revisited in light of recent structural data.

Nature has evolved different strategies for the reversible cleavage of ketose phosphosugars as essential metabolic reactions in all domains of life. Prominent examples are the Schiff-base forming class I FBP and F6P aldolase as well as transaldolase, which all exploit an active center lysine to reversibly cleave the C3-C4 bond of fructose-1,6-bisphosphate or fructose-6-phosphate to give two 3-carbon products (aldolase), or to shuttle 3-carbon units between various phosphosugars (transaldolase). In contrast, transketolase and phosphoketolase make use of the bioorganic cofactor thiamin diphosphate to cleave the preceding C2-C3 bond of ketose phosphates. While transketolase catalyzes the reversible transfer of 2-carbon ketol fragments in a reaction analogous to that of transaldolase, phosphoketolase forms acetyl phosphate as final product in a reaction that comprises ketol cleavage, dehydration and phosphorolysis. In this review, common and divergent catalytic principles of these enzymes will be discussed, mostly, but not exclusively, on the basis of crystallographic snapshots of catalysis. These studies in combination with mutagenesis and kinetic analysis not only delineated the stereochemical course of substrate binding and processing, but also identified key catalytic players acting at the various stages of the reaction. The structural basis for the different chemical fates and lifetimes of the central enamine intermediates in all five enzymes will be particularly discussed, in addition to the mechanisms of substrate cleavage, dehydration and ring-opening reactions of cyclic substrates. The observation of covalent enzymatic intermediates in hyperreactive conformations such as Schiff-bases with twisted double-bond linkages in transaldolase and physically distorted substrate-thiamin conjugates with elongated substrate bonds to be cleaved in transketolase, which probably epitomize a canonical feature of enzyme catalysis, will be also highlighted.

The transaldolase, a novel allergen of Fusarium proliferatum, demonstrates IgE cross-reactivity with its human analogue.

Fusarium species are among airborne fungi and recognized as causative agents of human atopic disorders. However, Fusarium allergens have not been well characterized and the lack of information limits clinical diagnosis and treatment of fungal allergy. The purpose of this study is to identify and characterize important allergens of F. proliferatum. IgE-reacting F. proliferatum components were identified by immunoblot using serum samples from patients of respiratory atopic diseases. Characterization of allergens and determination of IgE cross-reactivity were performed by cDNA cloning, then homologous expression and immunoblot inhibition studies. We identified nine different F. proliferatum components that can be recognized by IgE antibodies in 17 (28%) of the 60 atopic sera tested. Components with molecular masses of about 43, 37.5 and 36.5 kDa with IgE-binding frequencies of about 88, 47 and 53%, respectively, were considered as important allergens of F. proliferatum. The 37.5 kDa IgE-binding component was putatively considered as a transaldolase protein of F. proliferatum. The full-length cDNA of F. proliferatum transaldolase was subsequently cloned. It encodes an open reading frame of 312 amino acids and has sequence identifies of 73 and 61%, respectively, with Cladosporium and human transaldolases. The purified recombinant F. proliferatum transaldolase can inhibit the IgE-binding against the 37.5 kDa component of F. proliferatum and the transaldolase allergen from Cladosporium cladosporioides. More importantly, the recombinant F. proliferatum transaldolase can inhibit IgE-binding against human transaldolase in a concentration-dependent manner. Thus, a novel and important F. proliferatum transaldolase allergen was identified. In addition to IgE cross-reactivity between the Fusarium and the Cladosporium transaldolase allergens, IgE cross-reactivity between the Fusarium and the human transaldolases also exists and might contribute to atopic manifestations in the absence of exogenous allergen exposure.

Adherence to Bürgi-Dunitz stereochemical principles requires significant structural rearrangements in Schiff-base formation: insights from transaldolase complexes.

The Bürgi-Dunitz angle (αBD) describes the trajectory of approach of a nucleophile to an electrophile. The adoption of a stereoelectronically favorable αBD can necessitate significant reactive-group repositioning over the course of bond formation. In the context of enzyme catalysis, interactions with the protein constrain substrate rotation, which could necessitate structural transformations during bond formation. To probe this theoretical framework vis-à-vis biocatalysis, Schiff-base formation was analysed in Francisella tularensis transaldolase (TAL). Crystal structures of wild-type and Lys→Met mutant TAL in covalent and noncovalent complexes with fructose 6-phosphate and sedoheptulose 7-phosphate clarify the mechanism of catalysis and reveal that substrate keto moieties undergo significant conformational changes during Schiff-base formation. Structural changes compelled by the trajectory considerations discussed here bear relevance to bond formation in a variety of constrained enzymic/engineered systems and can inform the design of covalent therapeutics.

Structure-based gene targeting discovery of sphaerimicin, a bacterial translocase I inhibitor.

Rise and shine: Using a gene-targeting approach aimed at identifying potential L-threonine:uridine-5'-transaldolases that catalyze the formation of (5'S,6'S)-C-glycyluridine, a new bacterial translocase I inhibitor was discovered from an actinomycete following fermentation optimization.

Conservation of structure and mechanism within the transaldolase enzyme family.

Transaldolase (Tal) is involved in the central carbon metabolism, i.e. the non-oxidative pentose phosphate pathway, and is therefore a ubiquitous enzyme. However, Tals show a low degree in sequence identity and vary in length within the enzyme family which previously led to the definition of five subfamilies. We wondered how this variation is conserved in structure and function. To answer this question we characterised and compared the Tals from Bacillus subtilis, Corynebacterium glutamicum and Escherichia coli, each belonging to a different subfamily, with respect to their biochemical properties and structures. The overall structure of the Tal domain, a (ß/α)(8) -barrel fold, is well conserved between the different subfamilies but the enzymes show different degrees of oligomerisation (monomer, dimer and decamer). The substrate specificity of the three enzymes investigated is quite similar which is reflected in the conservation of the active site, the phosphate binding site as well as the position of a catalytically important water molecule. All decameric enzymes characterised so far appear to be heat stable no matter whether they originate from a mesophilic or thermophilic organism. Hence, the thermostability might be due to the structural properties, i.e. tight packing, of these enzymes. Database The crystal structures have been deposited in the Protein Data Bank with accession code 3R8R for BsTal and 3R5E for CgTal.

A thermostable recombinant transaldolase with high activity over a broad pH range.

Thermophilic enzymes are in high demand for various applications due to their prolonged lifetimes and high reaction rates at elevated temperatures. In this work, an open reading frame TM0295, which encodes a putative transaldolase (TAL) from a hyper-thermophilic microorganism, Thermotoga maritima, was cloned and expressed in Escherichia coli. The enzyme activity of transaldolase at high temperatures (e.g., at 80 °C) was reported here for the first time. The recombinant T. maritima transaldolase was extremely thermostable, with a half-life time of 198 and 13.0 h at 60 °C and 80 °C, respectively. The estimated total turn-over number was 1.5 × 10(6) mol of product per mol of enzyme at 80 °C. This enzyme also exhibited high activities within a broad pH range of 6.0-9.0. This ultra-thermostable TAL with high activity shows great potential for use in such applications as the production of enzymatic biofuels production and the synthesis of high-value carbohydrates by cell-free synthetic pathway biotransformation.

Twisted Schiff base intermediates and substrate locale revise transaldolase mechanism.

We examined the catalytic cycle of transaldolase (TAL) from Thermoplasma acidophilum by cryocrystallography and were able to structurally characterize--for the first time, to our knowledge--different genuine TAL reaction intermediates. These include the Schiff base adducts formed between the catalytic lysine and the donor ketose substrates fructose-6-phosphate and sedoheptulose-7-phosphate as well as the Michaelis complex with acceptor aldose erythrose-4-phosphate. These structural snapshots necessitate a revision of the accepted reaction mechanism with respect to functional roles of active site residues, and they further reveal fundamental insights into the general structural features of enzymatic Schiff base intermediates and the role of conformational dynamics in enzyme catalysis, substrate binding and discrimination. A nonplanar arrangement of the substituents around the Schiff base double bond was observed, suggesting that a structurally encoded reactant-state destabilization is a driving force of catalysis. Protein dynamics and the intrinsic hydrogen-bonding pattern appear to be crucial for selective recognition and binding of ketose as first substrate.

Crystallization and preliminary X-ray diffraction analysis of transaldolase from Thermoplasma acidophilum.

The metabolic enzyme transaldolase from Thermoplasma acidophilum was recombinantly expressed in Escherichia coli and could be crystallized in two polymorphic forms. Crystals were grown by the hanging-drop vapour-diffusion method using PEG 6000 as precipitant. Native data sets for crystal forms 1 and 2 were collected in-house to resolutions of 3.0 and 2.7 Å, respectively. Crystal form 1 belonged to the orthorhombic space group C222(1) with five monomers per asymmetric unit and crystal form 2 belonged to the monoclinic space group P2(1) with ten monomers per asymmetric unit.

The transaldolase family: new synthetic opportunities from an ancient enzyme scaffold.

Aldol reactions constitute a powerful methodology for carbon-carbon bond formation in synthetic organic chemistry. Biocatalytic carboligation by aldolases offers a green, uniquely regio- and stereoselective tool with which to perform these transformations. Recent advances in the field, fueled by both discovery and protein engineering, have greatly improved the synthetic opportunities for the atom-economic asymmetric synthesis of chiral molecules with potential pharmaceutical relevance. New aldolases derived from the transaldolase scaffold (based on transaldolase B and fructose-6-phosphate aldolase from Escherichia coli) have been shown to be unusually flexible in their substrate scope; this makes them particularly valuable for addressing an expanded molecular range of complex polyfunctional targets. Extensive knowledge arising from structural and molecular biochemical studies makes it possible to address the remaining limitations of the methodology by engineering tailored biocatalysts.

The characterization of transaldolase gene tal from Pichia stipitis and its heterologous expression in Fusarium oxysporum.

The 972 bp length of transaldolase gene tal was cloned from Pichia stipitis CICC1960, encoding a 323 amino acid protein with a calculated molecular mass of 35.36 kDa and isoelectric point of 5.20. Real time PCR analysis demonstrated that the mRNA transcript level of constitutive tal gene rise on xylose, glucose, fructose, mannose, galactose and sucrose as carbon source, respectively. Furthermore, the transcription of tal gene in P. stipitis on xylose was higher than on other carbon source, indicating that transaldolase plays a part in xylose utilization. To deeply study the tal gene biological function, it was expressed in Fusarium oxysporum CCTCC M209040. Recombinant transaldolase activity of transformant F. oxysporum M209040-Tal2 was about 0.52 U mg(-1) protein and was 1.57 times higher than that of the wild type F. oxysporum CCTCC M209040, indicating that the improvement of transaldolase activity in transformant was due to expression of the exogenous tal gene. Growth of transformant F. oxysporum M209040-Tal2 without selection pressure did not affect the level of hygromycin resistance of the transformants, suggesting that integrated tal gene was stable in mitosis. Fermentation trials of F. oxysporum M209040-Tal2 showed that the ethanol yield improved by 8.39 and 11.71% on glucose and xylose substrates, respectively, demonstrating that the expression of tal gene from P. stipitis CICC1960 in F. oxysporum CCTCC M209040 could improve ethanol production.

Redesigning the active site of transaldolase TalB from Escherichia coli: new variants with improved affinity towards nonphosphorylated substrates.

Recently, we reported on a transaldolase B variant (TalB F178Y) that is able to use dihydroxyacetone (DHA) as donor in aldol reactions. In a second round of protein engineering, we aimed at improving the affinity of this variant towards nonphosphorylated acceptor aldehydes, that is, glyceraldehyde (GA). The anion binding site was identified in the X-ray structure of TalB F178Y where a sulfate ion from the buffer was bound in the active site. Therefore, we performed site-directed saturation mutagenesis at three residues forming the putative phosphate binding site, Arg181, Ser226 and Arg228. The focused libraries were screened for the formation of D-fructose from DHA and d,l-GA by using an adjusted colour assay. The best results with respect to the synthesis of D-fructose were achieved with the TalB F178Y/R181E variant, which exhibited an at least fivefold increase in affinity towards d,l-GA (K(M)=24 mM). We demonstrated that this double mutant can use D-GA, glycolaldehyde and the L-isomer, L-GA, as acceptor substrates. This resulted in preparative synthesis of D-fructose, D-xylulose and L-sorbose when DHA was used as donor. Hence, we engineered a DHA-dependent aldolase that can synthesise the formation of polyhydroxylated compounds from simple and cheap substrates at preparative scale.