A standard numbering scheme for thiamine diphosphate-dependent decarboxylases

Abstract

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.

The Short‐chain Dehydrogenase/Reductase Engineering Database (SDRED): A classification and analysis system for a highly diverse enzyme family

“…A standard numbering scheme was established for Classical and Extended SDRs to facilitate the identification of structurally and functionally equivalent positions, as it had been previously shown for other enzyme families . Crystal structures were selected to cover mostly all homologous families of Classical and Extended SDRs in the SDRED with known PDB entries.…”

“…Protein family databases are useful tools to study the relationship between sequence, structure, and function of enzymes and to support the design of novel enzymes . In this study, the Short‐chain Dehydrogenase/Reductase Engineering Database ( https://sdred.biocatnet.de/ .)…”

Systematic Analysis of Large Enzyme Families: Identification of Specificity‐ and Selectivity‐Determining Hotspots

“…Thus, a numbering scheme for all decarboxylases was developed that assigns an identical number to each equivalent position in all members of the superfamily. [33] As a consequence, the residues of the S-pocket of the decarboxylases PfBAL, PpBFD, ApPDC, EcMenD, and EcAHAS II have the same standard number 477, although the respective amino acids and their numbers in the respective protein sequences differ (T481, L461, E469, F475, and V461, respectively). A profile-hidden Markov model was created by using a set of representative decarboxylases, including the pyruvate decarboxylase from S. cerevisiae as a reference sequence.…”

“…Upon aligning each decarboxylase sequence to the profile, the absolute residue numbering of the reference sequence was transferred to all members of the decarboxylase protein family. [33] By systematically comparing the sequences and structures of all decarboxylases, two hotspot positions were identified that mediate binding of the acceptor molecule: position 477 (standard numbering), which determines the size of the Spocket, and position 476, which might block the entrance to the S-pocket ( Table 1). …”

Navigating within thiamine diphosphate‐dependent decarboxylases: Sequences, structures, functional positions, and binding sites

“…On the other hand, the structural similarity of ThDP‐dependent decarboxylases, for instance, is not reflected at sequence level and even in the catalytic domains only comparably few positions are conserved . To facilitate the identification of functionally relevant amino acid positions in related enzymes, a standard numbering scheme was established for ThDP‐dependent decarboxylases using a profile hidden Markov model (HMM) derived from a structure‐guided multiple sequence alignment of representative sequences . The pyruvate decarboxylase from Saccharomyces cerevisiae (PDB accession 2VK8) was chosen as a reference for sequence numbering .…”