A Δ38 Deletion Variant of Human Transketolase as a Model of Transketolase-Like Protein 1 Exhibits No Enzymatic Activity

Abstract

Besides transketolase (TKT), a thiamin-dependent enzyme of the pentose phosphate pathway, the human genome encodes for two closely related transketolase-like proteins, which share a high sequence identity with TKT. Transketolase-like protein 1 (TKTL1) has been implicated in cancerogenesis as its cellular expression levels were reported to directly correlate with invasion efficiency of cancer cells and patient mortality. It has been proposed that TKTL1 exerts its function by catalyzing an unusual enzymatic reaction, a hypothesis that has been the subject of recent controversy. The most striking difference between TKTL1 and TKT is a deletion of 38 consecutive amino acids in the N-terminal domain of the former, which constitute part of the active site in authentic TKT. Our structural and sequence analysis suggested that TKTL1 might not possess transketolase activity. In order to test this hypothesis in the absence of a recombinant expression system for TKTL1 and resilient data on its biochemical properties, we have engineered and biochemically characterized a “pseudo-TKTL1” Δ38 deletion variant of human TKT (TKTΔ38) as a viable model of TKTL1. Although the isolated protein is properly folded under in vitro conditions, both thermal stability as well as stability of the TKT-specific homodimeric assembly are markedly reduced. Circular dichroism and NMR spectroscopic analysis further indicates that TKTΔ38 is unable to bind the thiamin cofactor in a specific manner, even at superphysiological concentrations. No transketolase activity of TKTΔ38 can be detected for conversion of physiological sugar substrates thus arguing against an intrinsically encoded enzymatic function of TKTL1 in tumor cell metabolism.

A key role for transketolase-like 1 in tumor metabolic reprogramming

“…Although our results suggest that TKTL1 affects cell metabolism via mechanisms that are either dependent or independent of its enzymatic activity, they also unambiguously show that TKTL1 has transketolase enzymatic activity, supporting previous reports [10, 20, 32], and refuting studies claiming that TKTL1 lacks enzymatic activity [39, 40]. In our studies, TKTL1 silencing lowered the overall cellular transketolase activity, supporting that TKTL1 cleaves a two-carbon fragment from a pentose-phosphate to generate GA3P.…”

Akt Phosphorylation and Regulation of Transketolase Is a Nodal Point for Amino Acid Control of Purine Synthesis

“…Two TKT-like proteins exist (TKTL1 and L2), but TKTL1 may be devoid of TKT activity and TKTL1 knockout mice have a mild phenotype (Schneider et al, 2012). TKT expression is up-regulated in various human cancers, and its activity correlates highly with tumor growth rate; TKT inhibitors reduce cancer cell proliferation in vitro and in vivo (Cascante et al, 2000; Comin-Anduix et al, 2001; Rais et al, 1999).…”

How does cancer cell metabolism affect tumor migration and invasion?

“…TKT, along with transaldolase (TAL), which transfers three-carbon units, a reversible connection between glycolysis, and the PPP. 102 A mutated transketolase transcript (TKTL1) is upregulated in human malignancies, and the overexpression of TKTL1 has been reported in different cancers. [103][104][105] TKTL1 is responsible for around 60% or 70% of transketolase activity in human hepatoma and colon-cancer cells.…”