Biomedical subjects

Natalia Nemeria

Publications and source records attributed to Natalia Nemeria.

11 recordsLinked to original sources

PubMed2006

The 1',4'-iminopyrimidine tautomer of thiamin diphosphate is poised for catalysis in asymmetric active centers on enzymes.

Thiamin diphosphate, a key coenzyme in sugar metabolism, is comprised of the thiazolium and 4'-aminopyrimidine aromatic rings, but only recently has participation of the 4'-aminopyrimidine moiety in catalysis gained wider acceptance. We report the use of electronic spectroscopy to identify the various tautomeric forms of the 4'-aminopyrimidine ring on four thiamin diphosphate enzymes, all of which decarboxylate pyruvate: the E1 component of human pyruvate dehydrogenase complex, the E1 subunit of Escherichia coli pyruvate dehydrogenase complex, yeast pyruvate decarboxylase, and pyruvate oxidase from Lactobacillus plantarum. It is shown that, according to circular dichroism spectroscopy, both the 1',4'-iminopyrimidine and the 4'-aminopyrimidine tautomers coexist on the E1 component of human pyruvate dehydrogenase complex and pyruvate oxidase. Because both tautomers are seen simultaneously, these two enzymes provide excellent evidence for nonidentical active centers (asymmetry) in solution in these multimeric enzymes. Asymmetry of active centers can also be induced upon addition of acetylphosphinate, an excellent electrostatic pyruvate mimic, which participates in an enzyme-catalyzed addition to form a stable adduct, resembling the common predecarboxylation thiamin-bound intermediate, which exists in its 1',4'-iminopyrimidine form. The identification of the 1',4'-iminopyrimidine tautomer on four enzymes is almost certainly applicable to all thiamin diphosphate enzymes: this tautomer is the intramolecular trigger to generate the reactive ylide/carbene at the thiazolium C2 position in the first fundamental step of thiamin catalysis.

Binding Sites↗

PubMed2006

Active-site changes in the pyruvate dehydrogenase multienzyme complex E1 apoenzyme component from Escherichia coli observed at 2.32 A resolution.

The first enzymatic component, E1 (EC 1.2.4.1), of the pyruvate dehydrogenase multienzyme complex (PDHc) utilizes thiamine diphosphate (ThDP) and Mg(2+) as cofactors. The structure of a branched-chain-specific E1 apoenzyme from the heterotetrameric alpha(2)beta(2) E1 family was recently reported and showed that disorder-to-order transformations in two active-site loops take place upon cofactor binding. To ascertain what effect the absence of cofactor may have in the homodimeric alpha(2) Escherichia coli PDHc E1, the corresponding apoenzyme has been prepared and its three-dimensional structure determined and analyzed at 2.32 A by crystallographic methods. This represents the first reported apoenzyme structure for any E1 component from the homodimeric alpha(2) family. Electron-density features occurring in the region where the cofactor pyrimidine ring would normally be expected to bind are of size, shape and location compatible with water molecules that form a hydrogen-bonded linkage between residues Glu571 and Val192, which normally make conserved interactions with the ThDP cofactor. A histidine side chain that normally forms hydrogen bonds to ThDP is disordered in its absence and partially occupies two sites. Unlike in the reported heterotetrameric branched-chain apo-E1, no disorder/order loop transformations are evident in apo-PDHc E1 relative to the holo-E1 enzyme (PDHc E1-ThDP-Mg(2+)). Differences in the extent of hydrogen-bonding networks found in the apo-E1 enzyme, the holo-E1 enzyme and in an inhibitor complex with bound thiamine 2-thiazolone diphosphate (ThTDP), PDHc E1-ThTDP-Mg(2+), are described.

Apoenzymes↗

PubMed2006

A thiamin-bound, pre-decarboxylation reaction intermediate analogue in the pyruvate dehydrogenase E1 subunit induces large scale disorder-to-order transformations in the enzyme and reveals novel structural features in the covalently bound adduct.

The crystal structure of the E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc) has been determined with phosphonolactylthiamin diphosphate (PLThDP) in its active site. PLThDP serves as a structural and electrostatic analogue of the natural intermediate alpha-lactylthiamin diphosphate (LThDP), in which the carboxylate from the natural substrate pyruvate is replaced by a phosphonate group. This represents the first example of an experimentally determined, three-dimensional structure of a thiamin diphosphate (ThDP)-dependent enzyme containing a covalently bound, pre-decarboxylation reaction intermediate analogue and should serve as a model for the corresponding intermediates in other ThDP-dependent decarboxylases. Regarding the PDHc-specific reaction, the presence of PLThDP induces large scale conformational changes in the enzyme. In conjunction with the E1-PLThDP and E1-ThDP structures, analysis of a H407A E1-PLThDP variant structure shows that an interaction between His-407 and PLThDP is essential for stabilization of two loop regions in the active site that are otherwise disordered in the absence of intermediate analogue. This ordering completes formation of the active site and creates a new ordered surface likely involved in interactions with the lipoyl domains of E2s within the PDHc complex. The tetrahedral intermediate analogue is tightly held in the active site through direct hydrogen bonds to residues His-407, Tyr-599, and His-640 and reveals a new, enzyme-induced, strain-related feature that appears to aid in the decarboxylation process. This feature is almost certainly present in all ThDP-dependent decarboxylases; thus its inclusion in our understanding of general thiamin catalysis is important.

Amino Acid Substitution↗

PubMed2005

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 K(d,ThDP) = 4.34 microM and K(m,ThDP) = 11 microM 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.

Acetolactate Synthase↗

PubMed2005

Evidence for dramatic acceleration of a C-H bond ionization rate in thiamin diphosphate enzymes by the protein environment.

The hypothesis that thiamin diphosphate-dependent enzymes achieve a significant fraction of their catalytic rate acceleration by providing a protein environment that helps to stabilize unstable zwitterionic/dipolar intermediates (including the enamine/C2alpha-carbanion present on all such enzymes) was tested experimentally using the intermediate C2alpha-hydroxyethylthiamin diphosphate (HEThDP) with the Escherichia coli pyruvate dehydrogenase complex and its E1 subunit (PDHc-E1). Using pre-steady-state and steady-state methods, it was shown that HEThDP is a substrate for this enzyme after ionization of the C2alpha-H bond. An experiment was then carried out to measure the PDHc-E1 catalyzed pre-steady-state rate constant for the D --> H exchange from the C2alpha position of HEThDP-d(4), as an indicator of the formation of the enamine. Importantly, the enzyme accelerates the rate of ionization of this bond by a factor of 10(7), corresponding to a 10 kcal/mol stabilization of the enamine intermediate by the enzyme. This finding is likely a general feature of thiamin diphosphate enzymes.

Acetylation↗

PubMed2004

Amino-terminal residues 1-45 of the Escherichia coli pyruvate dehydrogenase complex E1 subunit interact with the E2 subunit and are required for activity of the complex but not for reductive acetylation of the E2 subunit.

While N-terminal amino acids 1-55 are not seen in the structure of the Escherichia coli pyruvate dehydrogenase complex E1 subunit (PDHc-E1), mass spectrometric analysis indicated that this amino-terminal region of PDHc-E1 was protected by PDHc-E2. Hence, five deletion constructs of PDHc-E1 were created, Delta6-15, Delta16-25, Delta26-35, Delta36-45, and Delta46-55, along with single-site substitutions at Asp7, Asp9, Pro10, Ile11, Glu12, Thr13, Arg14, and Asp15. The decarboxylation of pyruvate and the ability of PDHc-E1 to dimerize are not affected by any of the deletions or substitutions. While Delta46-55 and the Pro10Ala, Ile11Ala, and Thr13Ala variants could form a complex with PDHc-E2, and produced NADH in the overall assay, Delta16-25, Delta26-35, and Delta36-45 and the Asp7Ala, Asp9Ala, Glu12Gln, Glu12Asp, Arg14Ala, and Asp15Ala variants failed in both respects. Remarkably, all constructs of PDHc-E1 from E. coli, as well as PDHc-E1 from Mycobacterium tuberculosis, could carry out reductive acetylation of the E. coli lipoyl domain, but only constructs of the E. coli PDHc-E1 could reductively acetylate E. coli PDHc-E2. It was concluded that there are at least two loci of interaction between the PDHc-E1 and PDHc-E2 subunits: (1) the thiamin diphosphate-bound substrate on PDHc-E1 and the lipoylamide of PDHc-E2, as reflected by the ability to reductively acetylate the latter; and (2) amino terminal residues 1-45 of PDHc-E1 with regions of PDHc-E2 (so far undefined for the E. coli complex), as reflected by the overall activity of the entire complex. These studies add important information regarding recognition within this multienzyme complex class with an alpha(2) E1 assembly.

Acetylation↗

PubMed2004

Tetrahedral intermediates in thiamin diphosphate-dependent decarboxylations exist as a 1',4'-imino tautomeric form of the coenzyme, unlike the michaelis complex or the free coenzyme.

Two circular dichroism signals observed on thiamin diphosphate (ThDP)-dependent enzymes, a positive band in the 300-305 nm range and a negative one in the 320-330 nm range, were investigated on yeast pyruvate decarboxylase (YPDC) and on the E1 subunit of the Escherichia coli pyruvate dehydrogenase complex (PDHc-E1). Addition of the tetrahedral ThDP-acetaldehyde adduct, 2-alpha-hydroxyethylThDP, to PDHc-E1 generates the positive band at 300 nm, consistent with the formation of the 1',4'-iminopyrimidine tautomer, as also demonstrated for phosphonolactylthiamin diphosphate, a stable analogue of the tetrahedral ThDP-pyruvate adduct 2-alpha-lactylThDP (Jordan, F. et al. (2003) J. Am. Chem. Soc. 125, 12732-12738). Therefore, we suggest that all tetrahedral ThDP-bound covalent complexes will also prefer this tautomer, and that the 4'-aminopyrimidine of ThDP participates in multiple steps of acid-base catalysis on ThDP enzymes. Studies with YPDC and PDHc-E1, and their active center variants, in conjunction with chemical models, enabled assignment of the negative band at 330 nm to a charge-transfer transition between the 4'-aminopyrimidine tautomer (presumed electron donor) and the thiazolium ring (presumed electron acceptor) of ThDP, with no significant contributions from any amino acid side chain of the proteins. However, in both YPDC and PDHc-E1, the presence of substrate or substrate surrogate was required to enable detection, suggesting that the band at 320-330 nm be used as a reporter for the Michaelis complex, involving the amino tautomer, on both enzymes. As the positive band near 300 nm reports on the 1',4'-imino tautomer of ThDP, methods are now available for kinetic monitoring of both tautomeric forms.

Binding Sites↗

PubMed2004

Structural determinants of enzyme binding affinity: the E1 component of pyruvate dehydrogenase from Escherichia coli in complex with the inhibitor thiamin thiazolone diphosphate.

Thiamin thiazolone diphosphate (ThTDP), a potent inhibitor of the E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc), binds to the enzyme with greater affinity than does the cofactor thiamin diphosphate (ThDP). To identify what determines this difference, the crystal structure of the apo PDHc E1 component complex with ThTDP and Mg(2+) has been determined at 2.1 A and compared to the known structure of the native holoenzyme, PDHc E1-ThDP-Mg(2+) complex. When ThTDP replaces ThDP, reorganization occurs in the protein structure in the vicinity of the active site involving positional and conformational changes in some amino acid residues, a change in the V coenzyme conformation, addition of new hydration sites, and elimination of others. These changes culminate in an increase in the number of hydrogen bonds to the protein, explaining the greater affinity of the apoenzyme for ThTDP. The observed hydrogen bonding pattern is not an invariant feature of ThDP-dependent enzymes but rather specific to this enzyme since the extra hydrogen bonds are made with nonconserved residues. Accordingly, these sequence-related hydrogen bonding differences likewise explain the wide variation in the affinities of different thiamin-dependent enzymes for ThTDP and ThDP. The sequence of each enzyme determines its ability to form hydrogen bonds to the inhibitor or cofactor. Mechanistic roles are suggested for the aforementioned reorganization and its reversal in PDHc E1 catalysis: to promote substrate binding and product release. This study also provides additional insight into the role of water in enzyme inhibition and catalysis.

Binding Sites↗

PubMed2003

Expression and purification of the dihydrolipoamide acetyltransferase and dihydrolipoamide dehydrogenase subunits of the Escherichia coli pyruvate dehydrogenase multienzyme complex: a mass spectrometric assay for reductive acetylation of dihydrolipoamide acetyltransferase.

Plasmids were constructed for overexpression of the Escherichia coli dihydrolipoamide acetyltransferase (1-lip E2, with a single hybrid lipoyl domain per subunit) and dihydrolipoamide dehydrogenase (E3). A purification protocol is presented that yields homogeneous recombinant 1-lip E2 and E3 proteins. The hybrid lipoyl domain was also expressed independently. Masses of 45,953+/-73Da (1-lip E2), 50,528+/-5.5Da (apo-E3), 51,266+/-48Da (E3 including FAD), and 8982+/-4.0 (lipoyl domain) were determined by MALDI-TOF mass spectrometry. The purified 1-lip E2 and E3 proteins were functionally active according to the overall PDHc activity measurement. The lipoyl domain was fully acetylated after just 30 s of incubation with E1 and pyruvate. The mass of the acetylated lipoyl domain is 9019+/-2Da according to MALDI-TOF mass spectrometry. Treatment of the 1-lip E2 subunit with trypsin resulted in the appearance of the lipoyl domain with a mass of 10,112+/-3Da. When preincubated with E1 and pyruvate, this tryptic fragment was acetylated according to the mass increase. MALDI-TOF mass spectrometry was thus demonstrated to be a fast and precise method for studying the reductive acetylation of the recombinant 1-lip E2 subunit by E1 and pyruvate.

Acetylation↗

PubMed2002

Histidine 407, a phantom residue in the E1 subunit of the Escherichia coli pyruvate dehydrogenase complex, activates reductive acetylation of lipoamide on the E2 subunit. An explanation for conservation of active sites between the E1 subunit and transketolase.

Least squares alignment of the E. coli pyruvate dehydrogenase multienzyme complex E1 subunit and yeast transketolase crystal structures indicates a general structural similarity between the two enzymes and provides a plausible location for a short-loop region in the E1 structure that was unobserved due to disorder. The residue H407, located in this region, is shown to be able to penetrate the active site. Suggested by this comparison, the H407A E1 variant was created, and H407 was shown to participate in the reductive acetylation of both an independently expressed lipoyl domain and the intact 1-lipoyl E2 subunit. While the H407A substitution only modestly affected the reaction through pyruvate decarboxylation (ca. 14% activity compared to parental E1), the overall complex has a much impaired activity, at most 0.15% compared to parental E1. Isothermal titration calorimetry measurements show that the binding of the lipoyl domain to the H407A E1 variant is much weaker than that to parental E1. At the same time, mass spectrometric measurements clearly demonstrate much impaired reductive acetylation of the independently expressed lipoyl domain and of the intact 1-lipoyl E2 by the H407A variant compared to the parental E1. A proposal is presented to explain the remarkable conservation of the three-dimensional structure at the active centers of the E. coli E1 subunit and transketolase on the basis of the parallels in the ligation-type reactions carried out and the need to protonate a very weak acid, a dithiolane sulfur atom in the former, and a carbonyl oxygen atom in the latter.

Acetylation↗

PubMed2002

Structure of the pyruvate dehydrogenase multienzyme complex E1 component from Escherichia coli at 1.85 A resolution.

The crystal structure of the recombinant thiamin diphosphate-dependent E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc) has been determined at a resolution of 1.85 A. The E. coli PDHc E1 component E1p is a homodimeric enzyme and crystallizes with an intact dimer in an asymmetric unit. Each E1p subunit consists of three domains: N-terminal, middle, and C-terminal, with all having alpha/beta folds. The functional dimer contains two catalytic centers located at the interface between subunits. The ThDP cofactors are bound in the "V" conformation in clefts between the two subunits (binding involves the N-terminal and middle domains), and there is a common ThDP binding fold. The cofactors are completely buried, as only the C2 atoms are accessible from solution through the active site clefts. Significant structural differences are observed between individual domains of E1p relative to heterotetrameric multienzyme complex E1 components operating on branched chain substrates. These differences may be responsible for reported alternative E1p binding modes to E2 components within the respective complexes. This paper represents the first structural example of a functional pyruvate dehydrogenase E1p component from any species. It also provides the first representative example for the entire family of homodimeric (alpha2) E1 multienzyme complex components, and should serve as a model for this class of enzymes.

Amino Acid Sequence↗