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The THIAMIN REQUIRING2 (TH2) protein comprising a mitochondrial targeting peptide followed by a transcription enhancement A and a haloacid dehalogenase domain is a thiamin monophosphate (TMP) phosphatase in the vitamin B1 biosynthetic pathway. The Arabidopsis th2-3 T-DNA insertion mutant was chlorotic and deficient in thiamin diphosphate (TDP). Complementation assays confirmed that haloacid dehalogenase domain alone was sufficient to rescue the th2-3 mutant. In pTH2:TH2-GFP/th2-3 complemented plants, the TH2-GFP was localized to the cytosol, mitochondrion, and nucleus, indicating that the vitamin B1 biosynthetic pathway extended across multi-subcellular compartments. Engineered TH2-GFP localized to the cytosol, mitochondrion, nucleus, and chloroplast, could complement the th2 mutant. Together, these results highlight the importance of intracellular TMP and thiamin trafficking in vitamin B1 biosynthesis. In an attempt to enhance the production of thiamin, we created various constructs to overexpress TH2-GFP in the cytosol, mitochondrion, chloroplast, and nucleus. Unexpectedly, overexpressing TH2-GFP resulted in an increase rather than a decrease in TMP. While studies on th2 mutants support TH2 as a TMP phosphatase, analyses of TH2-GFP overexpression lines implicating TH2 may also function as a TDP phosphatase in planta. We propose a working model that the TMP/TDP phosphatase activity of TH2 connects TMP, thiamin, and TDP into a metabolic cycle. The TMP phosphatase activity of TH2 is required for TDP biosynthesis, and the TDP phosphatase activity of TH2 may modulate TDP homeostasis in Arabidopsis.

Funder: Cambridge Trusts, Thiamine pyrophosphate (TPP), an essential co-factor for all species, is biosynthesised through a metabolically expensive pathway regulated by TPP riboswitches in bacteria, fungi, plants and green algae. Diatoms are microalgae responsible for c. 20% of global primary production. They have been predicted to contain TPP aptamers in the 3'UTR of some thiamine metabolism-related genes, but little information is known about their function and regulation. We used bioinformatics, antimetabolite growth assays, RT-qPCR, targeted mutagenesis and reporter constructs to test whether the predicted TPP riboswitches respond to thiamine supplementation in diatoms. Gene editing was used to investigate the functions of the genes with associated TPP riboswitches in Phaeodactylum tricornutum. We found that thiamine-related genes with putative TPP aptamers are not responsive to supplementation with thiamine or its precursor 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP), and targeted mutation of the TPP aptamer in the THIC gene encoding HMP-P synthase does not deregulate thiamine biosynthesis in P. tricornutum. Through genome editing we established that PtTHIC is essential for thiamine biosynthesis and another gene, PtSSSP, is necessary for thiamine uptake. Our results highlight the importance of experimentally testing bioinformatic aptamer predictions and provide new insights into the thiamine metabolism shaping the structure of marine microbial communities with global biogeochemical importance.

In the biosynthesis of menaquinone in bacteria, the thiamine diphosphate-dependent enzyme MenD catalyzes the decarboxylative carboligation of α-ketoglutarate and isochorismate to (1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxycyclohex-3-ene-1-carboxylate (SEPHCHC). The regioisomer of SEPHCHC, namely (1R,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxycyclohex-2-ene-1-carboxylate (iso-SEPHCHC), has been considered as a possible product, however, its existence has been doubtful due to a spontaneous elimination of pyruvate from SEPHCHC to 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC). In this work, the regioisomer iso-SEPHCHC was distinguished from SEPHCHC by liquid chromatography-tandem mass spectrometry. Iso-SEPHCHC was purified and identified by NMR spectroscopy. Just as SEPHCHC remained hidden as a MenD product for more than two decades, its regioisomer iso-SEPHCHC has remained until now.

The reliable design and prediction of enzyme promiscuity to access transformations not observed in nature remains a long-standing challenge. Herein, we present the first example of an intramolecular stereoselective Stetter reaction catalyzed by benzaldehyde lyase, guided by the rational structure screening of various ThDP-dependent enzymes using molecular dynamics (MD) simulations. After optimization, high productivity (up to 99 %) and stereoselectivity (up to 99:1 e.r.) for this novel enzyme function was achieved.

Recently, we detected a novel biomarker in human saliva called calcium-binding protein, spermatid-associated 1 (CABS1). CABS1 protein had previously been described only in testis, and little was known of its characteristics other than it was considered a structurally disordered protein. Levels of human CABS1 (hCABS1) in saliva correlate with stress, whereas smaller sized forms of hCABS1 in saliva are associated with resilience to stress. Interestingly, hCABS1 also has an anti-inflammatory peptide sequence near its carboxyl terminus, similar to that of a rat prohormone, submandibular rat 1. We performed phylogenetic and sequence analysis of hCABS1. We found that from 72 CABS1 sequences currently annotated in the National Center for Biotechnology Information protein database, only 14 contain the anti-inflammatory domain "TxIFELL," all of which are primates. We performed structural unfoldability analysis using PONDER and FoldIndex and discovered three domains that are highly disordered. Predictions of three-dimensional structure of hCABS1 using RaptorX, IonCom, and I-TASSER software agreed with these findings. Predicted neutrophil elastase cleavage density also correlated with hCABS1 regions of high structural disorder. Ligand binding prediction identified Ca2+ , Mg2+ , Zn2+ , leucine, and thiamine pyrophosphate, a pattern observed in enzymes associated with energy metabolism and mitochondrial localization. These new observations on hCABS1 raise intriguing questions about the interconnection between the autonomic nervous system, stress, and the immune system. However, the precise molecular mechanisms involved in the complex biology of hCABS1 remain unclear. We provide a detailed in silico analysis of relevant aspects of the structure and function of hCABS1 and postulate extracellular and intracellular roles.

The synthesis of complex molecules from simple, renewable carbon units is the goal of a sustainable economy. Here we explored the biocatalytic potential of the thiamine‐diphosphate‐dependent (ThDP) oxalyl‐CoA decarboxylase (OXC)/2‐hydroxyacyl‐CoA lyase (HACL) superfamily that naturally catalyzes the shortening of acyl‐CoA thioester substrates through the release of the C1‐unit formyl‐CoA. We show that the OXC/HACL superfamily contains promiscuous members that can be reversed to perform nucleophilic C1‐extensions of various aldehydes to yield the corresponding 2‐hydroxyacyl‐CoA thioesters. We improved the catalytic properties of Methylorubrum extorquens OXC by rational enzyme engineering and combined it with two newly described enzymes—a specific oxalyl‐CoA synthetase and a 2‐hydroxyacyl‐CoA thioesterase. This enzymatic cascade enabled continuous conversion of oxalate and aromatic aldehydes into valuable (S)‐α‐hydroxy acids with enantiomeric excess up to 99 %., The thiamine‐diphosphate‐dependent (ThDP) oxalyl‐CoA decarboxylase (OXC)/2‐hydroxyacyl‐CoA lyase (HACL) superfamily contains promiscuous members that can perform nucleophilic C1‐extensions of aldehydes to yield 2‐hydroxyacyl‐CoA thioesters. An enzymatic cascade consisting of OXC, oxalyl‐CoA synthetase, and a 2‐hydroxyacyl‐CoA thioesterase converted oxalate and aromatic aldehydes into (S)‐α‐hydroxy acids with enantiomeric excess up to 99 %.

The expression of Neurospora crassa NMT1 involved in thiamine pyrophosphate (TPP) metabolism is regulated at the level of mRNA splicing by a TPP-sensing riboswitch within the precursor NMT1 mRNA. Here, using the systematic helix-based computational method, we investigated the regulation of this riboswitch. We find that the function of the riboswitch does not depend on the transcription process. Whether TPP is present or not, the riboswitch predominately folds into the ON state, while the OFF state aptamer structure does not appear during transcription. Since the transition from the ON state to the aptamer structure is extremely slow, TPP may interact with the RNA before full formation of the aptamer structure, promoting the switch flipping. The potential to fully form helix P0 of the ON state is necessary to restore ligand-dependent gene control by the riboswitch.

Thiamine is a crucial cofactor involved in the maintenance of carbohydrate metabolism and participates in multiple cellular metabolic processes within the cytosol, mitochondria, and peroxisomes. Currently, four genetic defects have been described causing impairment of thiamine transport and metabolism: SLC19A2 dysfunction leads to diabetes mellitus, megaloblastic anemia and sensory-neural hearing loss, whereas SLC19A3, SLC25A19, and TPK1-related disorders result in recurrent encephalopathy, basal ganglia necrosis, generalized dystonia, severe disability, and early death. In order to achieve early diagnosis and treatment, biomarkers play an important role. SLC19A3 patients present a profound decrease of free-thiamine in cerebrospinal fluid (CSF) and fibroblasts. TPK1 patients show decreased concentrations of thiamine pyrophosphate in blood and muscle. Thiamine supplementation has been shown to improve diabetes and anemia control in Rogers' syndrome patients due to SLC19A2 deficiency. In a significant number of patients with SLC19A3, thiamine improves clinical outcome and survival, and prevents further metabolic crisis. In SLC25A19 and TPK1 defects, thiamine has also led to clinical stabilization in single cases. Moreover, thiamine supplementation leads to normal concentrations of free-thiamine in the CSF of SLC19A3 patients. Herein, we present a literature review of the current knowledge of the disease including related clinical phenotypes, treatment approaches, update of pathogenic variants, as well as in vitro and in vivo functional models that provide pathogenic evidence and propose mechanisms for thiamine deficiency in humans.

In recent years, (de)carboxylases that catalyze reversible (de)carboxylation have been targeted for application as carboxylation catalysts. This has led to the development of proof-of-concept (bio)synthetic CO2 fixation routes for chemical production. However, further progress towards industrial application has been hampered by the thermodynamic constraint that accompanies fixing CO2 to organic molecules. In this Review, biocatalytic carboxylation methods are discussed with emphases on the diverse strategies devised to alleviate the inherent thermodynamic constraints and their application in synthetic CO2 -fixation cascades.

Thiamine diphosphate (ThDP)-dependent enzymes catalyze a broad range of reactions with excellent enantioselectivity. Among these reactions, carboligations of aldehydes are of particular interest since the products, chiral hydroxy ketones, are valuable building blocks in the pharmaceutical industry. However, the substrates, for example, benzaldehyde, inactivate the biocatalysts, for example the ThDP-dependent benzaldehyde lyase from Pseudomonas fluorescens (PfBAL). Because only few mechanistic kinetic models for carboligation and simultaneous inactivation are available today, we quantitatively determined the reaction kinetics and inactivation of the self-carboligation of benzaldehyde yielding the product (R)-benzoin catalyzed by PfBAL directly from progress curves using model-based experimental analysis. Discrimination of several inactivation models identified the substrate-dependent inactivation by benzaldehyde to be significant. Sensitivity analysis and optimal experimental design improved parameter precision significantly, to between 4 and 26% relative standard deviation while maintaining the necessary number of 13 experiments moderate. The developed mechanistic kinetic model will enable to perform a model-based process optimization to circumvent the substrate-dependent enzyme inactivation. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018 © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1081-1092, 2018.

Multiple species and stocks of Pacific salmon Oncorhynchus spp. have experienced large declines in the number of returning adults over a wide region of the Pacific Northwest due to poor marine survival (low smolt-to-adult survival rates). One possible explanation for reduced survival is thiamine deficiency. Thiamine (vitamin B1 ) is an essential vitamin with an integral role in many metabolic processes, and thiamine deficiency is an important cause of salmonid mortality in the Baltic Sea and in the Laurentian Great Lakes. To assess this possibility, we (1) compared muscle thiamine content over time in a holding experiment using Fraser River (British Columbia) Sockeye Salmon O. nerka to establish whether adults that died during the holding period had lower thiamine levels than survivors, (2) measured infectious loads of multiple pathogens in held fish, and (3) measured egg thiamine content from four species of Pacific salmon collected on Fraser River spawning grounds. Chinook Salmon O. tshawytscha had the lowest egg thiamine, followed by Sockeye Salmon; however, egg thiamine concentrations were above levels known to cause overt fry mortality. Thiamine vitamers in the muscle of Fraser River adult Sockeye Salmon shifted over a 13-d holding period, with a precipitous decline in thiamine pyrophosphate (the active form of thiamine used in enzyme reactions) in surviving fish. Survivors also carried lower loads of Flavobacterium psychrophilum than fish that died during in the holding period. Although there is no evidence of thiamine deficiency in the adults studied, questions remain about possible thiamine metabolism-fish pathogen relationships that influence survival.

The catalytic asymmetric synthesis of chiral 2-hydroxy ketones by using different thiamine diphosphate dependent enzymes, namely benzaldehyde lyase from Pseudomonas fluorescens (PfBAL), a variant of benzoylformate decarboxylase from Pseudomonas putida (PpBFD-L461A), branched-chain 2-keto acid decarboxylase from Lactococcus lactis (LlKdcA) and a variant of pyruvate decarboxylase from Acetobacter pasteurianus (ApPDC-E469G), was studied. Starting with the same set of substrates, substituted benzaldehydes in combination with different aliphatic aldehydes, PfBAL and PpBFD-L461A selectively deliver the (R)- and (S)-2-hydroxy-propiophenone derivatives, respectively. The (R)- and (S)-phenylacetylcarbinol (1-hydroxy-1-phenylacetone) derivatives are accessible in a similar way using LlKdcA and ApPDC-E469G, respectively. In many cases excellent stereochemical purities (>98 % enantiomeric excess) could be achieved. Hence, the regio- and stereochemistry of the product in the asymmetric aliphatic–aromatic cross-benzoin reaction can be controlled solely by choice of the appropriate enzyme or enzyme variant.

Objective:The aim of this study was to investigate whether thiamine pyrophosphate (TPP) has biochemical and histological preventive effects on oxidative liver damage induced by paracetamol (APAP), in rats. Moreover,the additional benefits of TPP with N-acetylcysteine(NAC) were investigated. Methodology:Rats were divided into six groups.APAP 1500mg/kg orally, NAC and TPP at a dose of 100 mg/kg were administered intraperitoneally to the groups. Oxidant, antioxidant parameters and liver function tests were compared between groups. Additionally, the histological assessment of each group was performed.Results:In APAP group, a marked liver damage occured with the elevation of liver function tests and oxidative stress markers like MDA, MPO and NO. Simultaneously, biochemical results were congruent with the histological changes of oxidative damage. In the APAP+TPP group, the elevation of oxidants and the decrease in antioxidant levels like glutathione and catalase, were prevented by TPP. The histological changes were improved to almost normal hepatic structure, simultaneously. And the results showed that TPP and NAC have no additional benefits when used together.TPP and NAC had nearly similar hepatoprotective efficacy on APAP induced hepatotoxicity. Conclusion:As efficacious as the standard theraphy, TPP may be beneficial in APAP induced hepatotoxicity., Journal of International Society of Antioxidants in Nutrition & Health, Vol 3, No 1 (2016): Special Volume of ISANH Conference/Congress

Efficient production of D-lactate by engineered Escherichia coli entails balancing cell growth and product synthesis. To develop a metabolic switch to implement a desirable transition from cell growth to product fermentation, a thiamine auxotroph B0013-080A was constructed in a highly efficient D-lactate producer E. coli strain B0013-070. This was achieved by inactivation of thiE, a gene encoding a thiamine phosphate synthase for biosynthesis of thiamine monophosphate. The resultant mutant B0013-080A failed to grow on the medium in the absence of thiamine yet growth was restored when exogenous thiamine was provided. A linear relationship between cell mass formation and amount of thiamine supplemented was mathematically determined in a shake flask experiment and confirmed in a 7-L bioreactor system. This calculation revealed that ∼ 95-96 thiamine molecules per cell were required to satisfy cell growth. This relationship was employed to develop a novel fermentation process for D-lactate production by using thiamine as a limiting condition. A D-lactate productivity of 4.11 g · L(-1) · h(-1) from glycerol under microaerobic condition and 3.66 g · L(-1) · h(-1) from glucose under anaerobic condition was achieved which is 19.1% and 10.2% higher respectively than the parental strain. These results revealed a convenient and reliable method to control cell growth and improve D-lactate fermentation. This control strategy could be applied to other biotechnological processes that require optimal allocation of carbon between cell growth and product formation.

A new method for reversed phase HPLC determination of thiamine and its major in vivo phosphorylation products, thiamine monophosphate (TMP) and thiamine pyrophosphate (TPP), was developed using tetrabutylammonium hydroxide as the ion-pairing agent. The separation was performed on a Phenomenex Kinetex EVO C18 column with a gradient of a phosphate-buffered aqueous solution of the ion-pair reagent and methanol. The duty cycle for the assay was 13 min and pyrithiamine was successfully used as the internal standard for the first time in a thiamine HPLC measurement protocol. Detection of the fluorescence derivatives of the analytes as well as the IS allowed for lower detection limits in order to support biological applications in cell culture models. The linearity, sensitivity, specificity, accuracy and precision of the method were evaluated and met the requirements specified by the US Food and Drug Administration. The calibration curves proved to be linear and the method was validated over the range from 1.0–4000 nm for both cells and the media where complete recovery of the analytes was also achieved. Copyright © 2015 John Wiley & Sons, Ltd.

Thiamine pyrophosphate (TPP)-dependent enzymes are present in all domains of life, which share a cofactor with TPP. However, a comprehensive analysis of TPP-dependent enzymes from sequenced genomes has not been reported and little is known about the evolutionary dynamics of this gene family in plants. In this study, the evolutionary relationships of the TPP-dependent enzymes were investigated by using a comprehensive genome-wide phylogenetic analysis. A total of 103 plant TPP-dependent enzyme sequences were identified in 16 plant species. Phylogenetic analyses suggest that these plant TPP-dependent genes fell into three major groups, acetolactate synthase, pyruvate dehydrogenase, and 2-hydroxyphytanoyl-CoA lyase. Our results showed fewer TPP-dependent genes in plants than that in other living organisms, with many types of genes lost during evolution possibly because they do not largely influence plant function. Within the plant kingdom, seed plants have more TPP genes compared to non-seed basal plants, which probably imply that seed plants developed more TPP genes to adapt to their environment. The present study provides important information for understanding the evolution and function of the TPP-dependent gene family in plants.

Alaskan Husky encephalopathy (AHE(1) ) is a fatal brain disease associated with a mutation in SLC19A3.1 (c.624insTTGC, c.625C>A). This gene encodes for a thiamine transporter 2 with a predominately (CNS) central nervous system distribution. Considering that brain is particularly vulnerable to thiamine deficiency because of its reliance on thiamine pyrophosphate (TPP)-dependent metabolic pathways involved in energy metabolism and neurotransmitter synthesis, we characterized the impact of this mutation on thiamine status, brain bioenergetics and the contribution of oxidative stress to this phenotype. In silico modeling of the mutated transporter indicated a significant loss of alpha-helices resulting in a more open protein structure suggesting an impaired thiamine transport ability. The cerebral cortex and thalamus of affected dogs were severely deficient in TPP-dependent enzymes accompanied by decreases in mitochondrial mass and oxidative phosphorylation (OXPHOS) capacity, and increases in oxidative stress. These results along with the behavioral and pathological findings indicate that the phenotype associated with AHE is consistent with a brain-specific thiamine deficiency, leading to brain mitochondrial dysfunction and increased oxidative stress. While some of the biochemical deficits, neurobehavior and affected brain areas in AHE were shared by Wernicke's and Korsakoff's syndromes, several differences were noted likely arising from a tissue-specific vs. that from a whole-body thiamine deficiency.

Three supramolecular compounds with monophosphate or pyrophosphate esters of thiamine, namely, [TPPH ·CFSO ·HO] (1), [TMPH ·(CdCl) ·2HO] (2), and [TMPH ·NO ·2.5HO] (3) (TPP = thiamine pyrophosphate, TMP = thiamine monophosphate) were synthesized in methanol or water systems at room temperature. They were characterized by elemental analysis, IR spectrocopy, and single X-ray diffraction. Structural analysis shows that they are all supramolecular compounds and there are abundant interhydrogen and intrahydrogen bonds in the molecules. In addition, combined with the quantum mechanical calculations, their potential reaction activity and molecular electronic configuration were discussed.

Thiamine diphosphate dependent enzymes are well known for catalyzing the asymmetric synthesis of chiral α-hydroxy ketones from simple prochiral substrates. The steric and chemical properties of the enzyme active site define the product spectrum. Enzymes catalyzing the carboligation of aromatic aldehydes to (S)-benzoins have not so far been identified. We were able to close this gap by constructing a chimeric enzyme, which catalyzes the synthesis of various (S)-benzoins with excellent enantiomeric excess (>99 %) and very good conversion.

The sulphur atoms of sulphur-containing cofactors that are essential for numerous cellular functions in living organisms originate from L-cysteine via cysteine desulphurase (CSD) activity. However, many (hyper)thermophilic archaea, which thrive in solfataric fields and are positioned near the root of the evolutionary tree of life, lack CSD orthologues. The existence of CSD orthologues in a subset of (hyper)thermophilic archaea is of interest with respect to the evolution of sulphur-trafficking systems for the cofactors. This study demonstrates that the disruption of the csd gene of Thermococcus kodakarensis, a facultative elemental sulphur (S(0))-reducing hyperthermophilic archaeon, encoding Tk-CSD, conferred a growth defect evident only in the absence of S(0), and that growth can be restored by the addition of S(0), but not sulphide. We show that the csd gene is not required for biosynthesis of thiamine pyrophosphate or molybdopterin, irrespective of the presence or absence of S(0), but is necessary for iron-sulphur cluster biosynthesis in the absence of S(0). Recombinant form of Tk-CSD expressed in Escherichia coli was obtained and it was found to catalyse the desulphuration of L-cysteine. The obtained data suggest that hyperthermophiles might benefit from a capacity for CSD-dependent iron-sulphur cluster biogenesis, which allows them to thrive outside solfataric environments.

1-Deoxy-D-xylulose 5-phosphate (DXP) synthase catalyzes the formation of DXP from pyruvate and D-glyceraldehyde 3-phosphate (GraP) in a thiamin diphosphate-dependent manner, and is the first step in the essential pathway to isoprenoids in human pathogens. Understanding the mechanism of this unique enzyme is critical for developing new anti-infective agents that selectively target isoprenoid biosynthesis. The present study used mutagenesis and a combination of protein fluorescence, CD and kinetics experiments to investigate the roles of Arg420, Arg478 and Tyr392 in substrate binding and catalysis. The results support a random sequential, preferred order mechanism, and predict that Arg420 and Arg478 are involved in binding of the acceptor substrate, GraP. D-Glyceraldehyde, an alternative acceptor substrate lacking the phosphoryl group predicted to interact with Arg420 and Arg478, also accelerates decarboxylation of the predecarboxylation intermediate C2α-lactylthiamin diphosphate (LThDP) on DXP synthase, indicating that this binding interaction is not absolutely required, and that the hydroxyaldehyde sufficiently triggers decarboxylation. Unexpectedly, Tyr392 contributes to GraP affinity, and is not required for LThDP formation or its GraP-promoted decarboxylation. Time-resolved CD spectroscopy and NMR experiments indicate that LThDP is significantly stabilized on R420A and Y392F variants as compared with wild-type DXP synthase in the absence of acceptor substrate, but these substitutions do not appear to affect the rate of GraP-promoted LThDP decarboxylation in the presence of high levels of GraP, and LThDP formation remains the rate-limiting step. These results suggest a role of these residues in promoting GraP binding, which in turn facilitates decarboxylation, and also highlight interesting differences between DXP synthase and other thiamin diphosphate-dependent enzymes.

For almost 20 years, site-saturation mutagenesis (SSM) has been used to evolve stereoselective enzymes as catalysts for synthetic organic chemistry. Much of this work has focused on enzymes such as lipases and esterases, although the range is rapidly expanding. By contrast, using SSM to study enzyme mechanisms is much less common. Instead, site-directed mutagenesis is more generally employed, with a particular emphasis on alanine variants. In the present review, we provide examples of the growing use of SSM to study not only substrate and reaction selectivity, but also the reaction mechanism of thiamin diphosphate (ThDP)-dependent enzymes. We report that the use of SSM to examine the roles of the catalytic residues of benzoylformate decarboxylase gave rise to results that were at odds with earlier kinetic and structural studies using alanine substitutions and also questioned their conclusions. SSM was also employed to examine the long held tenet that a bulky hydrophobic residue provides a fulcrum by which the V-conformation of the ThDP cofactor is maintained. X-ray structures showed that ThDP stayed in the V-conformation even when the replacement residues were charged or did not contact the cofactor. We also summarize the results obtained when SSM was used to evolve new substrate specificity and/or enantioselectivity in ThDP-dependent enzymes such as benzoylformate decarboxylase, transketolase, 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate synthase and the E1 component of the 2-oxoglutarate dehydrogenase complex.

Kinetics and mechanism of photoprocesses generated by visible light-irradiation of the system riboflavin (Rf, vitamin B2) plus Thiamine (Th) and Thiamine pyrophosphate (ThDP), representing vitamin B1, was studied in pH 7 water. A weak dark complex vitamin B2-vitamin B1, with a mean value of 4 ± 0.4 M(-1) is formed. An intricate mechanism of competitive reactions operates upon photoirradiation, being the light only absorbed by Rf. Th and ThDP quench excited singlet and triplet states of Rf, with rate constants in the order of 10(9) and 10(6 ) M(-1 ) s(-1), respectively. With Vitamin B1 in a concentration similar to that of dissolved molecular oxygen in water, the quenching of triplet excited Rf by the latter is highly predominant, resulting in the generation of O(2)((1)Δ(g)). Superoxide radical anion was not detected under work conditions. A relatively slow O(2)((1)Δ(g))-mediated photodegradation of Th and ThDP was observed. Nevertheless, Th and especially ThDP behave as efficient physical deactivators of O(2)((1)Δ(g)). The thiazol structure in vitamin B1 appears as a good scavenger of this reactive oxygen species. This characteristic, that presents at vitamin B1 as a potential photoprotector of biological entities against O(2)((1)Δ(g)) attack, was been experimentally confirmed employing the protein lisozime as a photo-oxidizable target.

Calcium phosphate cements (CPC), forming hydroxyapatite during the setting reaction, are characterized by good biocompatibility and osteoconductivity, however, their remodeling into native bone tissue is slow. One strategy to improve remodeling and bone regeneration is the directed modification of their nanostructure. In this study, a CPC was set in the presence of cocarboxylase, glucuronic acid, tartaric acid, α-glucose-1-phosphate, L-arginine, L-aspartic acid, and L-lysine, respectively, with the aim to influence formation and growth of hydroxyapatite crystals through the functional groups of these biomolecules. Except for glucuronic acid, all these modifications resulted in the formation of smaller and more agglomerated hydroxyapatite particles which had a positive impact on the biological performance indicated by first experiments with the human osteoblast cell line hFOB 1.19. Moreover, adhesion, proliferation, and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSC) as well as binding of the growth factors BMP-2 and VEGF was investigated on CPC modified with cocarboxylase, arginine, and aspartic acid. Initial adhesion of hBMSC was improved on these three modifications and proliferation was enhanced on CPC modified with cocarboxylase and arginine whereas osteogenic differentiation remained unaffected. Modification of the CPC with arginine and aspartic acid, but not with cocarboxylase, led to a higher BMP-2 binding.

The crystal structure of Bifidobacterium longum phosphoketolase, a thiamine diphosphate (TPP) dependent enzyme, has been determined at 2.2Å resolution. The enzyme is a dimer with the active sites located at the interface between the two identical subunits with molecular mass of 92.5kDa. The bound TPP is almost completely shielded from solvent except for the catalytically important C2-carbon of the thiazolium ring, which can be accessed by a substrate sugar through a narrow funnel-shaped channel. In silico docking studies of B. longum phosphoketolase with its substrate enable us to propose a model for substrate binding.Structured summaryMINT-7985878: PKT (uniprotkb:Q6R2Q7) and PKT (uniprotkb:Q6R2Q7) bind (MI:0407) by X-ray crystallography (MI:0114)

Amish microcephaly (MCPHA, OMIM #607196) is a metabolic disorder that has been previously characterized by severe infantile lethal congenital microcephaly and alpha-ketoglutaric aciduria. All reported patients have been from the Pennsylvania Amish community and homozygous for a p.Gly177Ala mutation in SLC25A19. We present a further male patient with MCPHA born to distantly consanguineous parents in Ontario, Canada with Amish ancestors. Microcephaly was evident at 21 weeks gestation on ultrasound. At birth, the facial appearance and brain MRI scan were characteristic of MCPHA, with the additional features of partial agenesis of the corpus callosum and a closed spinal dysraphic state. Urine levels of alpha-ketoglutaric acid were normal at birth and during metabolic crisis, but were markedly elevated during a time of metabolic stability. A severe lactic acidosis was present during metabolic crises and responded to treatment with a high fat diet. At age 7 years, the child is healthy but has severe microcephaly and profound developmental delay. SLC25A19 has been described as a mitochondria inner membrane transporter for both deoxynucleotides and thiamine pyrophosphate (TPP). The biochemical phenotype of MCPHA may be attributable to decreased activity of the three mitochondrial enzymes that require TPP as a cofactor: pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, and branched chain amino acid dehydrogenase. We confirm that alpha-ketoglutaric aciduria is not a constant finding in MCPHA and suggest that a persistent lactic acidemia may be more common. The diagnosis should be considered in patients with severe congenital microcephaly, especially in association with lissencephaly, dysgenesis of the corpus callosum, or a spinal dysraphic state.

Summary The levels of transketolase (TK) and the amount of TK apoenzyme measured by thiamine pyrophosphate (TPP) addition in the erythrocytes of dairy cows, beef cattle and sheep are described. The TK levels were lower and the enhancement of the enzyme activity after TPP addition was greater in high yielding cows than in dry cows. A similar pattern was seen in beef cattle and sheep, with low TK levels and clearly enhanced enzyme activity after TPP addition in animals with clinical signs or suspected to have cerebrocortical necrosis (CCN). The possible presence of thiamine antagonists in the blood of these animals is also discussed. Zusammenfassung Transketolaseaktivitat im Blut von Rind und Schaf in Abhangigkeit von der Thiaminversorgung Bei Milchrindern, Fleischrindern und Schafen werden die Transketolaseaktivitaten (TK) und der Gehalt an TK-Apoenzym nach Hinzufugen von Thiaminpyrophosphat (TPP) beschrieben. Bei Kuhen mit hoher Milchleistung waren die TK-Spiegel niedriger und die Steigerung der Enzymaktivitat nach Hinzufugen von TPP ausgepragter als bei trockenstehenden Kuhen. Ein ahnliches Muster wurde bei Fleischrindern und Schafen beobachtet. Erythrozyten von Tieren mit Verdacht oder klinischen Anzeichen einer Cerebrocorticalnekrose wiesen niedrigere TK-Aktivitaten und deutlich erhohte Enzymaktivitaten nach Hinzufugen von TPP auf. Das mogliche Vorkommen eines Thiaminantagonisten im Blut dieser Tiere wird diskutiert. Resume Activite de la transketolase dans le sang de bovin et de mouton en fonction de l'approvisionnement en thiamine Les activites de la transketolase (TK) et le taux de TK-apoenzyme apres adjonction de pyrophosphate de thiamine (TPP) sont decrits chez des vaches laitieres, des bovins d'engrais et des moutons. Le taux TK fut plus bas et l'augmentation de l'activite enzymatique apres adjonction de TPP fut plus marque chez les vaches fortement laitieres que chez les vaches en periode de mise a goutte. On a observe un phenomene semblable chez des bovins d'engrais et des moutons. Des erythrocytes d'animaux suspects ou cliniquement atteints de necrose du cortex cerebral ont presente des activites TK plus basses et des activites enzymatiques nettement augmentees apres adjonction de TPP. On discute la presence possible d'un antagoniste de la thiamine dans le sang de ces animaux. Resumen La actividad transcetolasica en la sangre de vacunos y ovinos en relacion con la deficiencia de tiamina Se describen en vacas lecheras, vacunos destinados a carniceria y en ovinos las actividades de la transcetolasa (TC) y la tasa de apoenzima TC tras agregar pirofosfato de tiamina (PFT). En vacas con produccion elevada de leche eran menores los niveles de TC y mas evidente el incremento de la actividad enzimatica tras anadir PFT que en las vacas secas. Un modelo semejante fue observado en las reses para carne y en las ovejas. Los eritrocitos de animales con sospecha o signos clinicos de una necrosis cerebrocortical (NCC) presentaron actividades TC menores y actividades enzimaticas aumentadas con toda claridad despues de adicionar PFT. Discutese la presencia posible de un antagonista de la tiamina en la sangre de estos animales.

The mitochondrial carriers are a family of transport proteins that shuttle metabolites, nucleotides and cofactors across the inner mitochondrial membrane. The genome of Drosophila melanogaster encodes at least 46 members of this family. Only five of these have been characterized, whereas the transport functions of the remainder cannot be assessed with certainty. In the present study, we report the functional identification of two D. melanogaster genes distantly related to the human and yeast thiamine pyrophosphate carrier (TPC) genes as well as the corresponding expression pattern throughout development. Furthermore, the functional characterization of the D. melanogaster mitochondrial thiamine pyrophosphate carrier protein (DmTpc1p) is described. DmTpc1p was over-expressed in bacteria, the purified protein was reconstituted into liposomes, and its transport properties and kinetic parameters were characterized. Reconstituted DmTpc1p transports thiamine pyrophosphate and, to a lesser extent, pyrophosphate, ADP, ATP and other nucleotides. The expression of DmTpc1p in Saccharomyces cerevisiaeTPC1 null mutant abolishes the growth defect on fermentable carbon sources. The main role of DmTpc1p is to import thiamine pyrophosphate into mitochondria by exchange with intramitochondrial ATP and/or ADP.

After the recent discovery of bacterial riboswitches, synthetic riboswitches have been engineered by using natural and artificial RNA aptamers. In contrast to natural riboswitches, the majority of synthetic riboswitches in bacteria reported to date are ON switches that activate gene expression in response to the aptamer ligand. In this study, we adopted a mechanism-guided approach to design libraries predisposed to contain OFF riboswitches that respond to thiamine pyrophosphate (TPP). The first library design exploited a pseudo-Shine-Dalgarno (SD) sequence located near the 3'-end of the TPP aptamer, which would be less accessible to the ribosome when the aptamer is bound to TPP. In the second library, an SD sequence was strategically placed in the aptamer's P1 stem, which is stabilized upon ligand binding. OFF riboswitches were obtained by dual genetic selection of these libraries. The results underscore the importance of effective library design to achieve desired riboswitch functions.

Thiamin diphosphate-dependent enzymes participate in numerous biosynthetic pathways and catalyse a broad range of reactions, mainly involving the cleavage and formation of C-C bonds. For example, they catalyse the nonoxidative and oxidative decarboxylation of 2-keto acids, produce 2-hydroxy ketones and transfer activated aldehydes to a variety of acceptors. Moreover, they can also catalyse C-N, C-O and C-S bond formation. Because of their substrate spectra and different stereospecificity, these enzymes extend the synthetic potential for asymmetric carboligations appreciably. Different strategies have been developed to identify new members of this promiscuous enzyme class and the reactions they catalyse. This enabled us to introduce solutions for longstanding synthetic problems, such as asymmetric cross-benzoin condensation. Moreover, through a combination of protein structure analysis, enzyme and substrate engineering, and screening methods we explored additional stereochemical routes that have not been described previously for any of these interesting enzymes.

Prokaryotes, yeasts and plants synthesize thiamin (vitamin B1) via complex pathways. Animal cells capture the vitamin through specific high-affinity transporters essential for internal thiamin homeostasis. Inside the cells, thiamin is phosphorylated to higher phosphate derivatives. Thiamin diphosphate (ThDP) is the best-known thiamin compound because of its role as an enzymatic cofactor. However, in addition to ThDP, at least three other thiamin phosphates occur naturally in most cells: thiamin monophosphate, thiamin triphosphate (ThTP) and the recently discovered adenosine thiamin triphosphate. It has been suggested that ThTP has a specific neurophysiological role, but recent data favor a much more basic metabolic function. During amino acid starvation, Escherichia coli accumulate ThTP, possibly acting as a signal involved in the adaptation of the bacteria to changing nutritional conditions. In animal cells, ThTP can phosphorylate some proteins, but the physiological significance of this mechanism remains unknown. Adenosine thiamin triphosphate, recently discovered in E. coli, accumulates during carbon starvation and might act as an alarmone. Among the proteins involved in thiamin metabolism, thiamin transporters, thiamin pyrophosphokinase and a soluble 25-kDa thiamin triphosphatase have been characterized at the molecular level, in contrast to thiamin mono- and diphosphatases whose specificities remain to be proven. A soluble enzyme catalyzing the synthesis of adenosine thiamin triphosphate from ThDP and ADP or ATP has been partially characterized in E. coli, but the mechanism of ThTP synthesis remains elusive. The data reviewed here illustrate the complexity of thiamin biochemistry, which is not restricted to the cofactor role of ThDP.

Thiamine and its three phosphorylated derivatives (mono-, di- and triphosphate) occur naturally in most cells. Recently, we reported the presence of a fourth thiamine derivative, adenosine thiamine triphosphate, produced in Escherichia coli in response to carbon starvation. Here, we show that the chemical synthesis of adenosine thiamine triphosphate leads to another new compound, adenosine thiamine diphosphate, as a side product. The structure of both compounds was confirmed by MS analysis and H-1-, C-13- and P-31-NMR, and some of their chemical properties were determined. Our results show an upfield shifting of the C-2 proton of the thiazolium ring in adenosine thiamine derivatives compared with conventional thiamine phosphate derivatives. This modification of the electronic environment of the C-2 proton might be explained by a through-space interaction with the adenosine moiety, suggesting U-shaped folding of adenosine thiamine derivatives. Such a structure in which the C-2 proton is embedded in a closed conformation can be located using molecular modeling as an energy minimum. In E. coli, adenosine thiamine triphosphate may account for 15% of the total thiamine under energy stress. It is less abundant in eukaryotic organisms, but is consistently found in mammalian tissues and some cell lines. Using HPLC, we show for the first time that adenosine thiamine diphosphate may also occur in small amounts in E. coli and in vertebrate liver. The discovery of two natural thiamine adenine compounds further highlights the complexity and diversity of thiamine biochemistry, which is not restricted to the cofactor role of thiamine diphosphate.

A new large-scale purification method for benzoylformate decarboxylase from Pseudomonas putida has allowed us to undertake an X-ray crystallographic study of the enzyme. The previously observed instability of the enzyme was overcome by addition of 100 microM thiamine pyrophosphate to buffers used in the purification. The final enzyme preparation was more than 97% pure, as determined by denaturing gel electrophoresis and densitometry. The mobility of the enzyme on a gel filtration column indicates that it is a tetramer of 57-kDa subunits. Large, single crystals of benzoylformate decarboxylase were grown from solutions of buffered polyethylene glycol 400, pH 8.5. The crystals diffract to beyond 1.6 A resolution and are stable for days to X-ray radiation. Analysis of X-ray data from the crystals, along with the newly determined quaternary structure, identifies the space group as I222. The unit cell dimensions are a = 82 A, b = 97 A, c = 138 A. An average Vm value for the crystals is consistent with one subunit per asymmetric unit. The subunits of the tetramer must be arranged with tetrahedral 222 symmetry.

In the intestinal lumen thiamine is in free form and very low concentrations. Absorption takes place primarily in the proximal part of the small intestine by means of a dual mechanism, which is saturable at low (physiological) concentrations and diffusive at higher. Thiamine undergoes intracellular phosphorylation mainly to thiamine pyrophosphate, while at the serosal side only free thiamine is present. Thiamine uptake is enhanced by thiamine deficiency, and reduced by thyroid hormone and diabetes. The entry of thiamine into the enterocyte, as evaluated in brush border membrane vesicles of rat small intestine in the absence of H+ gradient, is Na+- and biotransformation-independent, completely inhibited by thiamine analogs and reduced by ethanol administration and aging. The transport involves a saturable mechanism at low concentrations of vitamin and simple diffusion at higher. Outwardly oriented H+ gradients enhance thiamine transport, whose saturable component is a Na+-independent electroneutral uphill process utilizing energy supplied by the H+ gradient, and involving a thiamine/ H+ 1:1 stoichiometric exchange. The exit of thiamine from the enterocyte, as evaluated in basolateral membrane vesicles, is Na+-dependent, directly coupled to ATP hydrolysis by Na+-K+-ATPase, and inhibited by thiamine analogs. Transport of thiamine by renal brush border membrane vesicles is similar to the intestinal as far as both H+ gradient influence and specificity are concerned. In the erythrocyte thiamine transport is a Na+-independent, electroneutral process yet with two components: saturable, prevailing at low thiamine concentrations, and diffusive at higher. The saturable (specific) component is missing in patients of the rare disease known as thiamine-responsive megaloblastic anaemia (TRMA), producing a general disturbance of thiamine transport up to thiamine deficiency. The TRMA gene is located in chromosome 1q23.3. Recently, the thiamine transporter has been cloned: it is a protein of 497 amino acid residues with high homology with the reduced-folate transporter.

Thiamin diphosphate (ThDP) is the biologically active form of vitamin B1 and an essential cofactor for a number of enzymes. The effect of solvent polarity on the tautomeric equilibria of ThDP using three model systems of the 4'-aminopyrimidine ring is studied by density functional theory calculations (B3LYP/6-311+G(d,p)//B3LYP/6-31G(d)) in the gas phase and selected solvents (cyclohexane, ether, dichloroethane, and water). Solvation effects are investigated using three different schemes: implicit solvation by a continuum model, explicit solvation by inclusion of three water molecules mimicking the first solvation shell of the enzymatic environment, and by a mixed implicit/explicit solvation model. The 4'-aminopyrimidine tautomer is more stable than the 1',4'-iminopyrimidine tautomer in all solvation schemes employed; however, the trend for the stabilities of the 1',4'-iminopyrimidine tautomer in the solvents depends on the specific ThDP-model. Formation of the catalytic important ylide for ThDP-dependent enzymes by deprotonation of ThDP(C2) is also investigated by localization of transition states for two possible pathways. Only the less stable tautomer, 1',4'-iminopyrimidine ThDP, is able to form the catalytic active ylide. Generation of the ylide through a direct intramolecular proton transfer from ThDP(C2) to the ThDP(N4') nitrogen lone pair is favored by 6 kcal/mol in the gas phase, as compared to a water-mediated ylide generation. However, inclusion of a dielectric medium reduces this difference dramatically. Furthermore, inclusion of two water molecules to model the apoenzymatic environment lowers the activation energies of both direct and water-mediated ylide generation.

Background: Thiamine status of ruminants is adversely affected in acidic rumen conditions. However, there have been limited published case study data related to thiamine deficiency of ruminants with acute ruminal lactic acidosis (ARLA). Hypothesis: Thiamine deficiency would occur in sheep with ARLA. Animals: Thirteen Ak-Karaman (white Karaman) sheep with ARLA, aged 1 year (ARLA group) and 10 healthy Ak-Karaman sheep, aged 1 year (control group) were used. Methods: After clinical examination, rumen fluid samples of all sheep were obtained with a stomach tube and examined immediately. Blood samples were taken from a jugular vein of the sheep. Erythrocytic transketolase enzyme activity and hence thiamine pyrophosphate (TPP) effect were determined according to Clausen's method. Results: History revealed that all sheep in the ARLA group had accidentally consumed excessive amounts of cracked barley. During clinical examination of the ARLA group, disturbed general condition, engorged scleral vessels, moderate to severe dehydration, and ruminal atony were recorded in the sheep. The results of the ruminal fluid analyses of the ARLA group demonstrated characteristics of ARLA. The results of clinical and ruminal fluid examination of control group were normal. The mean TPP effect (%) in the ARLA group (109 ± 28) was significantly higher than in the control group (22.2 ± 3.7) (P < .001). Conclusions and Clinical Importance: The present study revealed that severe thiamine deficiency occurred in sheep with ARLA. This result indicates that thiamine administration to sheep suffering from acute ruminal acidosis caused by overconsumption of readily fermentable carbohydrates could be beneficial in alleviating thiamine deficiency caused by ruminal acidosis.

Structural relationship within the family of the thiamine diphosphate-dependent 2-oxo acid dehydrogenases was analyzed by combining different methods of sequence alignment with crystallographic and enzymological studies of the family members. For the first time, the sequence similarity of the homodimeric 2-oxoglutarate dehydrogenase to heterotetrameric 2-oxo acid dehydrogenases is established. The presented alignment of the catalytic domains of the dehydrogenases of pyruvate, branched-chain 2-oxo acids and 2-oxoglutarate unravels the sequence markers of the substrate specificity and the essential residues of the family members without the 3D structures resolved. Predicted dual substrate specificity of some of the 2-oxo acid dehydrogenases was confirmed experimentally. The results were used to decipher functions of the two hypothetical proteins of animal genomes, OGDHL and DHTKD1, similar to the 2-oxoglutarate dehydrogenase. Conservation of all the essential residues confirmed their catalytic competence. Sequence analysis indicated that OGDHL represents a previously unknown isoform of the 2-oxoglutarate dehydrogenase, whereas DHTKD1 differs from the homologs at the N-terminus and substrate binding pocket. The differences suggest changes in heterologous protein interactions and accommodation of more polar and/or bulkier structural analogs of 2-oxoglutarate, such as 2-oxoadipate, 2-oxo-4-hydroxyglutarate, or products of the carboligase reaction between a 2-oxodicarboxylate and glyoxylate or acetaldehyde. The signatures of the Ca2+-binding sites were found in the Ca2+-activated 2-oxoglutarate dehydrogenase and OGDHL, but not in DHTKD1. Mitochondrial localization was predicted for OGDHL and DHTKD1, with DHTKD1 probably localized also to nuclei. Medical implications of the obtained results are discussed in view of the possible associations of the 2-oxo acid dehydrogenases and DHTKD1 with neurodegeneration and cancer. Proteins 2008. © 2007 Wiley-Liss, Inc.

Branched chain alpha-ketoacid dehydrogenase (BCKAD) deficiency, or maple syrup urine disease (MSUD), can be categorized as classical, intermediate, intermittent or thiamine responsive, based on generally concordant in vitro BCKAD activity and severity of phenotype. We present clinical and enzymatic data on a boy with intermediate maple syrup urine disease, and suggest that he represents a novel category of mutation. He presented at age 10 months in ketoacidotic coma, with a history of irritability, poor feeding and growth and developmental delay. Branched chain amino acid restriction effected normal growth and developmental parameters by age 42 months. In contrast to previous patients with intermediate MSUD, his fibroblasts and fibroblast extracts failed to decarboxylate [1-14C]-alpha-ketoisovalerate (KIV). The defect is not in mitochondrial transport of substrate, but rather in the catalytic activity of the E1 component of the BCKAD. Disrupted cells of the proband exhibited negligible BCKAD activity over a wide range of keto acid substrate concentrations, irrespective of the presence of added thiamine pyrophosphate (TPP). These results differ from the sigmoidal kinetics observed using classical MSUD extracts, and the hyperbolic kinetics with control preparations under the same assay conditions. We propose that the structurally altered enzyme possesses reduced but not negligible activity in vivo, and exists as an unstable complex in vitro under assay conditions used, even in the presence of added TPP.

Fibroblast strains derived from six patients with maple syrup urine disease have been investigated for their requirements of the cofactors NAD, CoASH, Mg++ and TPP in comparison with 10 normal control strains. The reconstitution of the decarboxylase function of branched chain alpha-keto acid (BCKA) dehydrogenase complex in lysed cells was studied with respect to the substrates alpha-keto-isocaproic acid, alpha-keto-isovaleric acid, and alpha-keto-beta-methylvaleric acid (KIC, KIVA, MEVA). The enzyme activity of all normal control strains for the substrates KIC and KIVA was not reconstituted by TPP + Mg++ alone, but CoASH + NAD could reconstitute the enzyme activity with KIC and KIVA in different degrees. Only two control strains were tested with MEVA as substrate, and these showed in contrast that TPP + Mg++ could partly reconstitute the enzyme activity. In contrast to the relative homogeneity in the reconstitution profiles of normal strains, the five classical and one intermittent MSUD strains showed heterogeneity in cofactor requirements. Complementation analysis using heterokaryons prepared from fibroblasts of four patients with classical MSUD and one patient with intermittent MSUD showed, in contrast to experiments with normal controls, a partial amelioration of the defect in two combinations; it is suggested that the defect in these strains is located at different functional subunits of the multienzyme complex.

Benzoylformate decarboxylase (BFD) from Pseudomonas putida is an exceptional thiamin diphosphate-dependent enzyme, as it catalyzes the formation of (S)-2-hydroxy-1-phenylpropan-1-one from benzaldehyde and acetaldehyde. This is the only currently known S-selective reaction (92 % ee) catalyzed by this otherwise R-selective class of enzymes. Here we describe the molecular basis of the introduction of S selectivity into ThDP-dependent decarboxylases. By shaping the active site of BFD through the use of rational protein design, structural analysis, and molecular modeling, optimal steric stabilization of the acceptor aldehyde in a structural element called the S pocket was identified as the predominant interaction for adjusting stereoselectivity. Our studies revealed Leu461 as a hot spot for stereoselectivity in BFD. Exchange to alanine and glycine resulted in variants that catalyze the S-stereoselective addition of larger acceptor aldehydes, such as propanal with benzaldehyde and its derivatives-a reaction not catalyzed by the wild-type enzyme. Crystal structure analysis of the variant BFDL461A supports the modeling studies.

Enzymes are biodegradable and renewable resources that utilize water as the reaction solvent. Reactions catalyzed by enzymes are among the most effective, selective, and ‘green’ processes available today. The advantages of using biocatalysts include their high degree of regio- and stereo-specificity, versatility, and high reaction rates under mild reaction conditions. Enzyme catalyzed enantioselective C–C bond formation reactions are rather important aspects of synthetic organic chemistry. Thiamine pyrophosphate (TPP) dependent enzymes, especially, have an important role in the stereoselective formation of C–C bonds. This review covers the recent advances in enzyme catalyzed asymmetric C–C bond formation using the most pronounced thiamine pyrophosphate dependent enzymes: acetohydroxyacid synthase, benzaldehyde lyase, benzoylformate decarboxylase, pyruvate decarboxylase, and phenylpyruvate decarboxylase. These enzymes are all capable of acyloin-type condensation reactions in water under mild conditions, in turn leading to chiral α-hydroxy ketones, which are versatile building blocks for the pharmaceutical and chemical industries.

Enzymatic carboligation in a solid/gas bioreactor represents a new challenge in biotechnology. In this paper, the continuous gas-phase production of propioin from two propanal molecules by using thiamine diphosphate-dependent enzymes was studied. Two enzymes were used, namely benzaldehyde lyase (BAL) from Pseudomonas fluorescens and benzoylformate decarboxylase (BFD) from Pseudomonas putida. The enzymes are homologous and catalyze carboligase and carbolyase reactions in which no external cofactor regeneration is needed. The influence of water and substrate activity on the initial reaction rate and biocatalyst stability was investigated. An increase in water activity raised the initial reaction rates to the maximal values of 250 and 80 U g(-1) for BAL and BFD, respectively. The half-life showed the same trend with maximal values of 50 and 78 min for BAL and BFD, respectively. The increase in the half-life by increasing water activity was unexpected. It was also observed that BFD is more stable than BAL in the presence of the substrate propanal. Both enzymes showed substrate inhibition in the kinetic studies, and BAL was also deactivated during the reaction. Unexpectedly, the stereoselectivity of both enzymes (ee of 19 % for BAL and racemic mixture for BFD) was significantly impaired in the gas phase compared to the liquid phase.

The interaction of thiamine diphosphate (ThDP) with transketolase (TK) involves at least two stages: [formula: see text] During the first stage, an inactive intermediate complex (TK...ThDP) is formed, which is then transformed into a catalytically active holoenzyme (TK* - ThDP). The second stage is related to conformational changes of the protein. In the preceding publication (Esakova, O. A., Meshalkina, L. E., Golbik, R., Hübner, G., and Kochetov, G. A. Eur. J. Biochem. 2004, 271, 4189 - 4194) we reported that the affinity of ThDP for TK considerably increases in the presence of the donor substrate, which may be a mechanism whereby the activity of the enzyme is regulated under the conditions of the coenzyme deficiency. Here, we demonstrate that the substrate affects the stage of the reverse conformational transition, characterized by the constant k(-1): in the presence of the substrate, its value is decreased several fold, whereas K(d) and k(+1) remain unchanged.