Danilo Meyer

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.

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.

Unexpected tautomeric equilibria of the carbanion-enamine intermediate in pyruvate oxidase highlight unrecognized chemical versatility of thiamin

Thiamin diphosphate, the vitamin B1 coenzyme, plays critical roles in fundamental metabolic pathways that require acyl carbanion equivalents. Studies on chemical models and enzymes had suggested that these carbanions are resonance-stabilized as enamines. A crystal structure of this intermediate in pyruvate oxidase at 1.1 Å resolution now challenges this paradigm by revealing that the enamine does not accumulate. Instead, the intermediate samples between the ketone and the carbanion both interlocked in a tautomeric equilibrium. Formation of the keto tautomer is associated with a loss of aromaticity of the cofactor. The alternate confinement of electrons to neighboring atoms rather than π-conjugation seems to be of importance for the enzyme-catalyzed, redox-coupled acyl transfer to phosphate, which requires a dramatic inversion of polarity of the reacting substrate carbon in two subsequent catalytic steps. The ability to oscillate between a nucleophilic (carbanion) and an electrophilic (ketone) substrate center highlights a hitherto unrecognized versatility of the thiamin cofactor. It remains to be studied whether formation of the keto tautomer is a general feature of all thiamin enzymes, as it could provide for stable storage of the carbanion state, or whether this feature represents a specific trait of thiamin oxidases. In addition, the protonation state of the two-electron reduced flavin cofactor can be fully assigned, demonstrating the power of high-resolution cryocrystallography for elucidation of enzymatic mechanisms.

Observation of a stable carbene at the active site of a thiamin enzyme

Carbenes are highly reactive chemical compounds that are exploited as ligands in organometallic chemistry and are powerful organic catalysts. They were postulated to occur as transient intermediates in enzymes, yet their existence in a biological system could never be demonstrated directly. We present spectroscopic and structural data of a thiamin enzyme in a noncovalent complex with substrate, which implicate accumulation of a stable carbene as a major resonance contributor to deprotonated thiamin.

Tuning and Switching Enantioselectivity of Asymmetric Carboligation in an Enzyme through Mutational Analysis of a Single Hot Spot

Enantioselective bond making and breaking is a hallmark of enzyme action, yet switching the enantioselectivity of the reaction is a difficult undertaking, and typically requires extensive screening of mutant libraries and multiple mutations. Here, we demonstrate that mutational diversification of a single catalytic hot spot in the enzyme pyruvate decarboxylase gives access to both enantiomers of acyloins acetoin and phenylacetylcarbinol, important pharmaceutical precursors, in the case of acetoin even starting from the unselective wild‐type protein. Protein crystallography was used to rationalize these findings and to propose a mechanistic model of how enantioselectivity is controlled. In a broader context, our studies highlight the efficiency of mechanism‐inspired and structure‐guided rational protein design for enhancing and switching enantioselectivity of enzymatic reactions, by systematically exploring the biocatalytic potential of a single hot spot.

A δ38 deletion variant of human transketolase as a model of transketolase-like protein 1 exhibits no enzymatic activity.

Besides transketolase (TKT), a thiamin-dependent enzyme of the pentose phosphate pathway, the human genome encodes for two closely related transketolase-like proteins, which share a high sequence identity with TKT. Transketolase-like protein 1 (TKTL1) has been implicated in cancerogenesis as its cellular expression levels were reported to directly correlate with invasion efficiency of cancer cells and patient mortality. It has been proposed that TKTL1 exerts its function by catalyzing an unusual enzymatic reaction, a hypothesis that has been the subject of recent controversy. The most striking difference between TKTL1 and TKT is a deletion of 38 consecutive amino acids in the N-terminal domain of the former, which constitute part of the active site in authentic TKT. Our structural and sequence analysis suggested that TKTL1 might not possess transketolase activity. In order to test this hypothesis in the absence of a recombinant expression system for TKTL1 and resilient data on its biochemical properties, we have engineered and biochemically characterized a “pseudo-TKTL1” Δ38 deletion variant of human TKT (TKTΔ38) as a viable model of TKTL1. Although the isolated protein is properly folded under in vitro conditions, both thermal stability as well as stability of the TKT-specific homodimeric assembly are markedly reduced. Circular dichroism and NMR spectroscopic analysis further indicates that TKTΔ38 is unable to bind the thiamin cofactor in a specific manner, even at superphysiological concentrations. No transketolase activity of TKTΔ38 can be detected for conversion of physiological sugar substrates thus arguing against an intrinsically encoded enzymatic function of TKTL1 in tumor cell metabolism.

Alternating Sites Reactivity Is a Common Feature of Thiamin Diphosphate- Dependent Enzymes As Evidenced by Isothermal Titration Calorimetry Studies of Substrate Binding

Thiamin diphosphate (ThDP)-dependent enzymes play vital roles in cellular metabolism in all kingdoms of life. In previous kinetic and structural studies, a communication between the active centers in terms of a negative cooperativity had been suggested for some but not all ThDP enzymes, which typically operate as functional dimers. To further underline this hypothesis and to test its universality, we investigated the binding of substrate analogue methyl acetylphosphonate (MAP) to three different ThDP-dependent enzymes acting on substrate pyruvate, namely, the Escherichia coli E1 component of the pyruvate dehydrogenase complex, E. coli acetohydroxyacid synthase isoenzyme I, and the Lactobacillus plantarum pyruvate oxidase using isothermal titration calorimetry. The results unambiguously show for all three enzymes studied that only one active center of the functional dimers accomplishes covalent binding of the substrate analogue, supporting the proposed alternating sites reactivity as a common feature of all ThDP enzymes and resolving the recent controversy in the field.

Degradation of lipoxygenase-derived oxylipins by glyoxysomes from sunflower and cucumber cotyledons.

BackgroundOilseed germination is characterized by the degradation of storage lipids. It may proceed either via the direct action of a triacylglycerol lipase, or in certain plant species via a specific lipid body 13-lipoxygenase. For the involvement of a lipoxygenase previous results suggested that the hydroxy- or oxo-group that is being introduced into the fatty acid backbone by this lipoxygenase forms a barrier to continuous β-oxidation.ResultsThis study shows however that a complete degradation of oxygenated fatty acids is possible by isolated cucumber and sunflower glyoxysomes. Interestingly, degradation is accompanied by the formation of saturated short chain acyl-CoAs with chain length between 4 and 12 carbon atoms lacking the hydroxy- or oxo-diene system of the oxygenated fatty acid substrate. The presence of these CoA esters suggests the involvement of a specific reduction of the diene system at a chain length of 12 carbon atoms including conversion of the hydroxy-group at C7.ConclusionsTo our knowledge this metabolic pathway has not been described for the degradation of polyunsaturated fatty acids so far. It may represent a new principle to degrade oxygenated fatty acid derivatives formed by lipoxygenases or chemical oxidation initiated by reactive oxygen species.