Molecular Evolutionary Analysis of the Thiamine-Diphosphate-Dependent Enzyme, Transketolase

Abstract

Abstract. Members of the transketolase group of thiamine-diphosphate-dependent enzymes from 17 different organisms including mammals, yeast, bacteria, and plants have been used for phylogenetic reconstruction. Alignment of the amino acid and DNA sequences for 21 transketolase enzymes and one putative transketolase reveals a number of highly conserved regions and invariant residues that are of predicted importance for enzyme activity, based on the crystal structure of yeast transketolase. One particular sequence of 36 residues has some similarities to the nucleotide-binding motif and we designate it as the transketolase motif. We report further evidence that the recP protein from Streptococcus pneumoniae might be a transketolase and we list a number of invariant residues which might be involved in substrate binding. Phylogenies derived from the nucleotide and the amino acid sequences by various methods show a conventional clustering for mammalian, plant, and gramnegative bacterial transketolases. The branching order of the gram-positive bacteria could not be inferred reliably. The formaldehyde transketolase (sometimes known as dihydroxyacetone synthase) of the yeast Hansenula polymorpha appears to be orthologous to the mammalian enzymes but paralogous to the other yeast transketolases. The occurrence of more than one transketolase gene in some organisms is consistent with several gene duplications. The high degree of similarity in functionally important residues and the fact that the same kinetic mechanism is applicable to all characterized transketolase enzymes is consistent with the proposition that they are all derived from one common ancestral gene. Transketolase appears to be an ancient enzyme that has evolved slowly and might serve as a model for a molecular clock, at least within the mammalian clade.

Heterologous expression of human transketolase

“…Furthermore, except for three synonymous substitutions, it is identical to the consensus sequence derived from the TK cDNA of controls and WK patients [17]. This sequence comparison further con®rms that the sequence submitted to GenBank by Singleton and collaborators contains four nonsynonymous substitutions, an observation which is supported by an alignment including TK sequences from rodents [23].…”

“…The coding region comprises 1869 nucleotides which are translated into a protein of 623 amino acid residues with a calculated subunit molecular weight of 67.8 kDa. Comparison of TK sequences from various species have revealed two regions of high homology [23]. The ®rst region, termed the ThDP-binding motif [11], is located between amino acid residues 154 and 177 and is found in all known ThDP-dependent enzymes.…”

“…The second region, located between residues 415 and 450, displays the highest degree of sequence similarity and has been shown to be characteristic of TK. This motif has been designated the TK motif [23].…”

“…catalysis, subunit interaction, substrate and cofactor binding. A detailed protein sequence comparison between most known TKs has shown that particular residues are conserved [23]. However, while bacterial, fungal and plant sequences could readily be aligned, major gaps had to be introduced into the mammalian sequences to allow alignment [23].…”

Properties and functions of the thiamin diphosphate dependent enzyme transketolase

“…The sequences are taken from the multiple alignment of 22 TK sequences in Schenk et al [138]. Only four sequences representing mammalian (Hsa: human [138]), plant (Cpl3: Craterostigma plantagineum [4]), fungal (Sce: Saccharomyces cerevisiae [155]) and bacterial (Eco: Escherischia coli [152]) sources have been selected for illustration. The consensus sequence based on the entire alignment in Schenk et al [138] is shown; x can be any amino acid.…”