Kai Tittmann

The crystal structure of human transketolase and new insights into its mode of action.

The crystal structure of human transketolase (TKT), a thiamine diphosphate (ThDP) and Ca2+-dependent enzyme that catalyzes the interketol transfer between ketoses and aldoses as part of the pentose phosphate pathway, has been determined to 1.75 Å resolution. The recombinantly produced protein crystallized in space group C2 containing one monomer in the asymmetric unit. Two monomers form the homodimeric biological assembly with two identical active sites at the dimer interface. Although the protomer exhibits the typical three (α/β)-domain structure and topology reported for TKTs from other species, structural differences are observed for several loop regions and the linker that connects the PP and Pyr domain. The cofactor and substrate binding sites of human TKT bear high resemblance to those of other TKTs but also feature unique properties, including two lysines and a serine that interact with the β-phosphate of ThDP. Furthermore, Gln189 spans over the thiazolium moiety of ThDP and replaces an isoleucine found in most non-mammalian TKTs. The side chain of Gln428 forms a hydrogen bond with the 4′-amino group of ThDP and replaces a histidine that is invariant in all non-mammalian TKTs. All other amino acids involved in substrate binding and catalysis are strictly conserved. Besides a steady-state kinetic analysis, microscopic equilibria of the donor half-reaction were characterized by an NMR-based intermediate analysis. These studies reveal that formation of the central 1,2-dihydroxyethyl-ThDP carbanion-enamine intermediate is thermodynamically favored with increasing carbon chain length of the donor ketose substrate. Based on the structure of human transketolase and sequence alignments, putative functional properties of the related transketolase-like proteins TKTL1 and -2 are discussed in light of recent findings suggesting that TKTL1 plays a role in cancerogenesis.

Conversion of Pyruvate Decarboxylase into an Enantioselective Carboligase with Biosynthetic Potential

Pyruvate decarboxylase (PDC) catalyzes the decarboxylation of pyruvate into acetaldehyde and CO(2) and requires the cofactors thiamin diphosphate and Mg(2+) for activity. Owing to its catalytic promiscuity and relaxed substrate specificity, PDC catalyzes carboligation side reactions and is exploited for the asymmetric synthesis of 2-hydroxy ketones such as (R)-phenylacetyl carbinol, the precursor of (-)-ephedrine. Although PDC variants with enhanced carboligation efficiency were generated in the past, the native reaction, i.e., formation of aldehydes, is heavily favored over carboligation side reactions in all these biocatalysts. We characterized an active site variant (Glu473Gln) in which partitioning between aldehyde release versus carboligation is inverted with an up to 100-fold preference for the latter pathway. Due to a defective protonation of the central carbanion/enamine intermediate, substrate turnover stalls at this catalytic stage and addition of external aldehydes leads to quantitative and enantioselective formation of 2-hydroxy ketones as shown for (R)-phenylacetyl carbinol, which is afforded with unmatched yields, rates, and purity. This protein variant thus constitutes an example for the rational design of biocatalysts with greatly enhanced accidental catalytic promiscuity by selective blockage of the native reaction and accumulation of reactive intermediates under steady-state turnover conditions.

Identification, Characterization, and Structure Analysis of the Cyclic di-AMP-binding PII-like Signal Transduction Protein DarA

Background: Cyclic di-AMP is an essential second messenger in eubacteria. Results: The c-di-AMP receptor DarA was identified in B. subtilis. The crystal structure and ITC data revealed the nucleotide specificity of DarA. Conclusion: DarA is a PII-like protein that undergoes conformational changes upon c-di-AMP binding. Significance: A novel PII-like protein is involved in c-di-AMP signaling. The cyclic dimeric AMP nucleotide c-di-AMP is an essential second messenger in Bacillus subtilis. We have identified the protein DarA as one of the prominent c-di-AMP receptors in B. subtilis. Crystal structure analysis shows that DarA is highly homologous to PII signal transducer proteins. In contrast to PII proteins, the functionally important B- and T-loops are swapped with respect to their size. DarA is a homotrimer that binds three molecules of c-di-AMP, each in a pocket located between two subunits. We demonstrate that DarA is capable to bind c-di-AMP and with lower affinity cyclic GMP-AMP (3′3′-cGAMP) but not c-di-GMP or 2′3′-cGAMP. Consistently the crystal structure shows that within the ligand-binding pocket only one adenine is highly specifically recognized, whereas the pocket for the other adenine appears to be promiscuous. Comparison with a homologous ligand-free DarA structure reveals that c-di-AMP binding is accompanied by conformational changes of both the fold and the position of the B-loop in DarA.

Reaction mechanisms of thiamin diphosphate enzymes: redox reactions.

Amongst a wide variety of different biochemical reactions in cellular carbon metabolism, thiamin diphosphate‐dependent enzymes catalyze the oxidative decarboxylation of 2‐keto acids. This type of reaction typically involves redox coupled acyl transfer to CoA or phosphate and is mediated by additional cofactors, such as flavins, iron‐sulfur clusters or lipoamide swinging arms, which transmit the reducing equivalents that arise during keto acid oxidation to a final electron acceptor. EPR spectroscopic and kinetic studies have implicated the intermediacy of radical cofactor intermediates in pyruvate:ferredoxin oxidoreductase and an acetyl phosphate‐producing pyruvate oxidase, whereas the occurrence of transient on‐pathway radicals in other enzymes is less clear. The structures of pyruvate:ferredoxin oxidoreductase and pyruvate oxidase with different enzymic reaction intermediates along the pathway including a radical intermediate were determined by cryo‐crystallography and used to infer electron tunneling pathways and the potential roles of CoA and phosphate for an intimate coupling of electron and acyl group transfer. Viable mechanisms of reductive acetylation in pyruvate dehydrogenase multi‐enzyme complex, and of electron transfer in the peripheral membrane enzyme pyruvate oxidase from Escherichia coli, are also discussed.

The inhibition mechanism of human 20S proteasomes enables next-generation inhibitor design

Insights into proteasome inhibition Proteasomes are large protein complexes that degrade and remove proteins to maintain proper cellular physiology and growth. Proteasomes are a validated target for anticancer therapy, but drug design has been hampered by poor understanding of how inhibitors interact with the active site. Schrader et al. succeeded in crystallizing various proteasome-inhibitor complexes. They subsequently obtained crystal structures for the native human proteasome and eight different inhibitor complexes at resolutions between 1.9 and 2.1 Å. The inhibitors sampled include drugs that are approved or in trial for cancer treatment. Science, this issue p. 594 High-resolution structures of human 20S proteasomes reveal chemical principles for next-generation drug design. The proteasome is a validated target for anticancer therapy, and proteasome inhibition is employed in the clinic for the treatment of tumors and hematological malignancies. Here, we describe crystal structures of the native human 20S proteasome and its complexes with inhibitors, which either are drugs approved for cancer treatment or are in clinical trials. The structure of the native human 20S proteasome was determined at an unprecedented resolution of 1.8 angstroms. Additionally, six inhibitor-proteasome complex structures were elucidated at resolutions between 1.9 and 2.1 angstroms. Collectively, the high-resolution structures provide new insights into the catalytic mechanisms of inhibition and necessitate a revised description of the proteasome active site. Knowledge about inhibition mechanisms provides insights into peptide hydrolysis and can guide strategies for the development of next-generation proteasome-based cancer therapeutics.

Control of the Diadenylate Cyclase CdaS in Bacillus subtilis AN AUTOINHIBITORY DOMAIN LIMITS CYCLIC DI-AMP PRODUCTION

Background: Bacillus subtilis CdaS is a sporulation-specific diadenylate cyclase. Results: Activity of CdaS is regulated by its N-terminal autoinhibitory domain. Conclusion: The synthesis of c-di-AMP is under tight control in B. subtilis. Significance: The activity of CdaS is governed by a hexamer/dimer transition. The Gram-positive bacterium Bacillus subtilis encodes three diadenylate cyclases that synthesize the essential signaling nucleotide cyclic di-AMP. The activities of the vegetative enzymes DisA and CdaA are controlled by protein-protein interactions with their conserved partner proteins. Here, we have analyzed the regulation of the unique sporulation-specific diadenylate cyclase CdaS. Very low expression of CdaS as the single diadenylate cyclase resulted in the appearance of spontaneous suppressor mutations. Several of these mutations in the cdaS gene affected the N-terminal domain of CdaS. The corresponding CdaS mutant proteins exhibited a significantly increased enzymatic activity. The N-terminal domain of CdaS consists of two α-helices and is attached to the C-terminal catalytically active diadenylate cyclase (DAC) domain. Deletion of the first or both helices resulted also in strongly increased activity indicating that the N-terminal domain serves to limit the enzyme activity of the DAC domain. The structure of YojJ, a protein highly similar to CdaS, indicates that the protein forms hexamers that are incompatible with enzymatic activity of the DAC domains. In contrast, the mutations and the deletions of the N-terminal domain result in conformational changes that lead to highly increased enzymatic activity. Although the full-length CdaS protein was found to form hexamers, a truncated version with a deletion of the first N-terminal helix formed dimers with high enzyme activity. To assess the role of CdaS in sporulation, we assayed the germination of wild type and cdaS mutant spores. The results indicate that cyclic di-AMP formed by CdaS is required for efficient germination.

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.

Valine 375 and phenylalanine 109 confer affinity and specificity for pyruvate as donor substrate in acetohydroxy acid synthase isozyme II from Escherichia coli.

Acetohydroxy acid synthase (AHAS) is a thiamin diphosphate-dependent enzyme that catalyzes the condensation of pyruvate with either another pyruvate molecule (product acetolactate) or 2-ketobutyrate (product acetohydroxybutyrate) as the first common step in the biosynthesis of branched-chain amino acids in plants, bacteria, algae, and fungi. AHAS isozyme II from Escherichia coli exhibits a 60-fold higher specificity for 2-ketobutyrate (2-KB) over pyruvate as acceptor, which was shown to result from a stronger hydrophobic interaction of the ethyl substituent of 2-KB with the side chain of Trp464 in multiple, apparently committed steps of catalysis. Here, we have elucidated the molecular determinants conferring specificity for pyruvate as the sole physiological donor substrate. Structural studies and sequence alignments of the POX subfamily of ThDP enzymes that act on pyruvate indicate that a valine and a phenylalanine hydrophobically interact with the methyl substituent of pyruvate. Kinetic and thermodynamic studies on AHAS isozyme II variants with substitutions at these positions (Val375Ala, Val375Ile, and Phe109Met) were carried out. While Val375 variants exhibit a slightly reduced k(cat) with a moderate increase of the apparent K(M) of pyruvate, both substrate affinity and k(cat) are significantly compromised in AHAS Phe109Met. The specificity for 2-ketobutyrate as acceptor is not altered in the variants. Binding of acylphosphonates as analogues of donor substrates was analyzed by circular dichroism spectroscopy and stopped-flow kinetics. While binding of the pyruvate analogue is 10-100-fold compromised in all variants, Val375Ala binds the 2-KB analogue better than the wild type and with higher affinity than the pyruvate analogue, suggesting steric constraints imposed by Val375 as a major determinant for the thermodynamically favored binding of pyruvate in AHAS. NMR-based intermediate analysis at steady state reveals that a mutation of either Val375 or Phe109 is detrimental for unimolecular catalytic steps in which tetrahedral intermediates are involved, such as substrate addition to the cofactor and product liberation. This observation implies Val375 and Phe109 to not only conjointly mediate substrate binding and specificity but moreover to ensure a proper orientation of the donor substrate and intermediates for correct orbital alignment in multiple transition states.

Glutamate 636 of the Escherichia coli pyruvate dehydrogenase-E1 participates in active center communication and behaves as an engineered acetolactate synthase with unusual stereoselectivity.

The residue Glu636 is located near the thiamine diphosphate (ThDP) binding site of the Escherichia coli pyruvate dehydrogenase complex E1 subunit (PDHc-E1), and to probe its function two variants, E636A and E636Q were created with specific activities of 2.5 and 26% compared with parental PDHc-E1. According to both fluorescence binding and kinetic assays, the E636A variant behaved according to half-of-the-sites mechanism with respect to ThDP. In contrast, with the E636Q variant a Kd,ThDP = 4.34 μm and Km,ThDP = 11 μm were obtained with behavior more reminiscent of the parental enzyme. The CD spectra of both variants gave evidence for formation of the 1′,4′-iminopyrimidine tautomer on binding of phosphonolactylthiamine diphosphate, a stable analog of the substrate-ThDP covalent complex. Rapid formation of optically active (R)-acetolactate by both variants, but not by the parental enzyme, was observed by CD and NMR spectroscopy. The acetolactate configuration produced by the Glu636 variants is opposite that produced by the enzyme acetolactate synthase and the Asp28-substituted variants of yeast pyruvate decarboxylase, suggesting that the active centers of the two sets of enzymes exhibit different facial selectivity (re or si) vis à vis pyruvate. The tryptic peptide map (mass spectral analysis) revealed that the Glu636 substitution changed the mobility of a loop comprising amino acid residues from the ThDP binding fold. Apparently, the residue Glu636 has important functions both in active center communication and in protecting the active center from undesirable “carboligase” side reactions.

Activation of thiamin diphosphate and FAD in the phosphatedependent pyruvate oxidase from Lactobacillus plantarum.

The phosphate- and oxygen-dependent pyruvate oxidase from Lactobacillus plantarum is a homotetrameric enzyme that binds 1 FAD and 1 thiamine diphosphate per subunit. A kinetic analysis of the partial reactions in the overall oxidative conversion of pyruvate to acetyl phosphate and CO2 shows an indirect activation of the thiamine diphosphate by FAD that is mediated by the protein moiety. The rate constant of the initial step, the deprotonation of C2-H of thiamine diphosphate, increases 10-fold in the binary apoenzyme-thiamine diphosphate complex to 10−2 s−1. Acceleration of this step beyond the observed overall catalytic rate constant to 20 s−1 requires enzyme-bound FAD. FAD appears to bind in a two-step mechanism. The primarily bound form allows formation of hydroxyethylthiamine diphosphate but not the transfer of electrons from this intermediate to O2. This intermediate form can be mimicked using 5-deaza-FAD, which is inactive toward O2 but active in an assay using 2,6-dichlorophenolindophenol as electron acceptor. This analogue also promotes the rate constant of C2-H dissociation of thiamine diphosphate in pyruvate oxidase beyond the overall enzyme turnover. Formation of the catalytically competent FAD-thiamine-pyruvate oxidase ternary complex requires a second step, which was detected at low temperature.