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

Abstract

Thiamine diphosphate‐dependent decarboxylases catalyze both cleavage and formation of CC bonds in various reactions, which have been assigned to different homologous sequence families. This work compares 53 ThDP‐dependent decarboxylases with known crystal structures. Both sequence and structural information were analyzed synergistically and data were analyzed for global and local properties by means of statistical approaches (principle component analysis and principal coordinate analysis) enabling complexity reduction. The different results obtained both locally and globally, that is, individual positions compared with the overall protein sequence or structure, revealed challenges in the assignment of separated homologous families. The methods applied herein support the comparison of enzyme families and the identification of functionally relevant positions. The findings for the family of ThDP‐dependent decarboxylases underline that global sequence identity alone is not sufficient to distinguish enzyme function. Instead, local sequence similarity, defined by comparisons of structurally equivalent positions, allows for a better navigation within several groups of homologous enzymes. The differentiation between homologous sequences is further enhanced by taking structural information into account, such as BioGPS analysis of the active site properties or pairwise structural superimpositions. The methods applied herein are expected to be transferrable to other enzyme families, to facilitate family assignments for homologous protein sequences.

Ancestral sequences of a large promiscuous enzyme family correspond to bridges in sequence space in a network representation

“…Protein sequence networks could be assessed further to elucidate the evolvability of substrate ambiguity or promiscuity as starting points for protein design studies [24,32,68]. The alignment of functional protein domains or selected active site positions could further facilitate the selection of interesting enzyme candidates [60] and the design of highly enriched, minimal mutant libraries.…”

“…The identification of functionally relevant amino acid positions for a certain (sub)family would require structural information on the active site, too. Various approaches have been reported previously for other protein families, for instance thiamine diphosphate-dependent decarboxylases [60] or cytochrome P450 monooxygenases [61]. Hubs are nodes with a high degree centrality in a network.…”

Decarboxylation in Natural Products Biosynthesis

“…However, attempts to modify substrate specificity through rational design approaches, such as residue swapping, have yielded limited success. 5,8 Further research is needed to uncover the mechanisms that determine how TPP-dependent decarboxylases recognize and interact with their substrates.…”

“…4 In heterolytic decarboxylation, a carbanion is formed at the α-carbon center where the C-CO 2 ̅ bond is broken. Stabilization of the carbanion requires an electron sink, often in the form of a cofactor like thiamine pyrophosphate (TPP), 5 pyridoxal 5′-phosphate (PLP), 6 or functional groups on the substrate that can stabilize the carbanion through conjugation such as ketone, thial, or pquinone methide. While most decarboxylases follow a heterolytic route, some enzymes break the C-CO 2 ̅ bond homolytically, resulting in radical intermediates during decarboxylation.…”

“…(B) General catalytic mechanism of TPP-dependent decarboxylases.Due to significant variations in domain organization among various TPP-dependent decarboxylase superfamilies, the specific placement of the catalytic Glu residue is not illustrated, and general bases are utilized in the mechanism. For a more extensive understanding of the structural and catalytic features of TPPdependent decarboxylases, we suggest referring to comprehensive review articles 5,8.…”