Covalently Bound Substrate at the Regulatory Site of Yeast Pyruvate Decarboxylases Triggers Allosteric Enzyme Activation

Kutter, Steffen; Weiss, M.S.; Wille, Georg; Golbik, Ralph; Spinka, Michael; König, Stephan

doi:10.1074/jbc.m806228200

Cited by 43 publications

(48 citation statements)

References 51 publications

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“…One of the representative proteins, the pyruvate decarboxylase from S . cerevisiae ( Sc PDC, Swissprot: P06169, PDB: 2VK8 [6]), was used as the reference sequence for numbering all proteins of the decarboxylase superfamily. In addition, 22 functionally and structurally relevant residues in the sequence of Sc PDC were annotated as described in literature [2,5,28,30,31,37-39] (Additional file 1: Table S1).…”

Section: Resultsmentioning

confidence: 99%

“…Because some members of the decarboxylase superfamily show activation upon binding of a substrate at a second (allosteric) binding site (e.g. Sc PDC) [6] which leads to conformational changes, the set of reference proteins only contained decarboxylases which show no substrate activation or which have been crystallized in complex with an allosteric activator. Thus, only structures of active enzymes were compared.…”

Section: Discussionmentioning

confidence: 99%

“…In contrast to the previously mentioned motif, this region is not part of the active site but is presumably relevant for the structure or regulation of the protein. The adjacent loop region 286–304 was described as a part of the activation cascade of pyruvate decarboxylases, since this loop shows structural rearrangement upon binding of an activator at the effector binding site at standard position 221 [6]. …”

Section: Discussionmentioning

confidence: 99%

“…They are separated by a third domain, which is less conserved and adopts different functions in the various enzyme families, e.g. by binding additional cofactors such as ADP [4] and FAD [5] or activators and inhibitors [6]. Due to structural relations between this middle domain and the transhydrogenase domain dIII, this domain is called the TH3 domain [2,3].…”

Section: Introductionmentioning

confidence: 99%

“…In this work, we present the establishment of a numbering scheme for the ThDP-dependent decarboxylases based on the sequence of the well-documented pyruvate decarboxylase from S . cerevisiae (PDB: 2VK8 [6], Swissprot: P06169). The numbering scheme was validated by comparing its ability to produce multisequence alignments to the T-Coffee alignment algorithm and by revision of the structural equivalence of positions with the same standard numbers.…”

Section: Introductionmentioning

confidence: 99%

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A standard numbering scheme for thiamine diphosphate-dependent decarboxylases

et al. 2012

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BackgroundStandard numbering schemes for families of homologous proteins allow for the unambiguous identification of functionally and structurally relevant residues, to communicate results on mutations, and to systematically analyse sequence-function relationships in protein families. Standard numbering schemes have been successfully implemented for several protein families, including lactamases and antibodies, whereas a numbering scheme for the structural family of thiamine-diphosphate (ThDP) -dependent decarboxylases, a large subfamily of the class of ThDP-dependent enzymes encompassing pyruvate-, benzoylformate-, 2-oxo acid-, indolpyruvate- and phenylpyruvate decarboxylases, benzaldehyde lyase, acetohydroxyacid synthases and 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate synthase (MenD) is still missing.Despite a high structural similarity between the members of the ThDP-dependent decarboxylases, their sequences are diverse and make a pairwise sequence comparison of protein family members difficult.ResultsWe developed and validated a standard numbering scheme for the family of ThDP-dependent decarboxylases. A profile hidden Markov model (HMM) was created using a set of representative sequences from the family of ThDP-dependent decarboxylases. The pyruvate decarboxylase from S. cerevisiae (PDB: 2VK8) was chosen as a reference because it is a well characterized enzyme. The crystal structure with the PDB identifier 2VK8 encompasses the structure of the ScPDC mutant E477Q, the cofactors ThDP and Mg2+ as well as the substrate analogue (2S)-2-hydroxypropanoic acid. The absolute numbering of this reference sequence was transferred to all members of the ThDP-dependent decarboxylase protein family. Subsequently, the numbering scheme was integrated into the already established Thiamine-diphosphate dependent Enzyme Engineering Database (TEED) and was used to systematically analyze functionally and structurally relevant positions in the superfamily of ThDP-dependent decarboxylases.ConclusionsThe numbering scheme serves as a tool for the reliable sequence alignment of ThDP-dependent decarboxylases and the unambiguous identification and communication of corresponding positions. Thus, it is the basis for the systematic and automated analysis of sequence-encoded properties such as structural and functional relevance of amino acid positions, because the analysis of conserved positions, the identification of correlated mutations and the determination of subfamily specific amino acid distributions depend on reliable multisequence alignments and the unambiguous identification of the alignment columns. The method is reliable and robust and can easily be adapted to further protein families.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A standard numbering scheme for thiamine diphosphate-dependent decarboxylases

et al. 2012

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S‐Selective Mixed Carboligation by Structure‐Based Design of the Pyruvate Decarboxylase from Acetobacter pasteurianus

et al. 2011

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The thiamine diphosphate (ThDP)‐dependent pyruvate decarboxylase from Acetobacter pasteurianus (ApPDC) catalyzes the carboligation of aldehydes that yields (R)‐2‐hydroxy ketones with high chemoselectivity in mixed carboligations of aliphatic donor and aromatic acceptor aldehydes. On the basis of the crystal structure of ApPDC, which was determined to a resolution of 2.75 Å, and biochemical data, we mapped the active site. This enabled us to design variants with tailor‐made catalytic activities by modifications of the residues E469 and W388. Although the exchange of W388 by smaller amino acids yields variants with higher carboligase activity due to an increased access to the active site, the exchange of E469 to glycine opens the so‐called S‐pocket in ApPDC for aromatic aldehydes and thus alters the stereoselectivity. The variant ApPDC‐E469G provides access to (S)‐phenylacetylcarbinol derivatives by enzymatic carboligation with a good stereoselectivity of up to 89 % enantiomeric excess. The variant nicely complements the toolbox of ThDP‐dependent enzymes, which now gives access to all stereo‐ and regioisomers of the asymmetric aliphatic–aromatic cross‐benzoin‐like condensation. We prove that optimal stabilization of both aldehydes in the active site is essential to gain high yields and high selectivities.

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Characterization of cysteine thiol modifications based on protein microenvironments and local secondary structures

Bhatnagar

Bandyopadhyay

2017

Proteins

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We have demonstrated earlier that protein microenvironments were conserved around disulfide-bridged cystine motifs with similar functions, irrespective of diversity in protein sequences. Here, cysteine thiol modifications were characterized based on protein microenvironments, secondary structures and specific protein functions. Protein microenvironment around an amino acid was defined as the summation of hydrophobic contributions from the surrounding protein fragments and the solvent molecules present within its first contact shell. Cysteine functions (modifications) were grouped into enzymatic and non-enzymatic classes. Modifications studied were-disulfide formation, thio-ether formation, metal-binding, nitrosylation, acylation, selenylation, glutathionylation, sulfenylation, and ribosylation. 1079 enzymatic proteins were reported from high-resolution crystal structures. Protein microenvironments around cysteine thiol, derived from above crystal structures, were clustered into 3 groups-buried-hydrophobic, intermediate and exposed-hydrophilic clusters. Characterization of cysteine functions were statistically meaningful for 4 modifications (disulfide formation, thioether formation, sulfenylation, and iron/zinc binding) those have sufficient amount of data in the current dataset. Results showed that protein microenvironment, secondary structure and protein functions were conserved for enzymatic cysteine functions, in contrast to the same function from non-enzymatic cysteines. Disulfide forming enzymatic cysteines were tightly packed within intermediate protein microenvironment cluster, have alpha-helical conformation and mostly belonged to CxxC motif of electron transport proteins. Disulfide forming non-enzymatic cysteines did not belong to conserved motif and have variable secondary structures. Similarly, enzymatic thioether forming cysteines have conserved microenvironment compared to non-enzymatic cystienes. Based on the compatibility between protein microenvironment and cysteine modifications, more efficient drug molecules could be designed against cysteine-related diseases.

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Covalently Bound Substrate at the Regulatory Site of Yeast Pyruvate Decarboxylases Triggers Allosteric Enzyme Activation

Cited by 43 publications

References 51 publications

A standard numbering scheme for thiamine diphosphate-dependent decarboxylases

A standard numbering scheme for thiamine diphosphate-dependent decarboxylases

S‐Selective Mixed Carboligation by Structure‐Based Design of the Pyruvate Decarboxylase from Acetobacter pasteurianus

Characterization of cysteine thiol modifications based on protein microenvironments and local secondary structures

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