Isolation and Biochemical Characterization of Recombinant Transketolase from Mycobacterium tuberculosis

Abstract

Transketolase, an enzyme of the pentose phosphate pathway, plays an important role in the functioning of mycobacteria. Using plasmid pET-19b carrying the Rv1449c gene of transketolase from Mycobacterium tuberculosis and an additional histidine tag, we isolated and purified recombinant transketolase and determined the conditions for obtaining the apoform of the protein. The Michaelis constants were evaluated for the thiamine diphosphate cofactor in the presence of magnesium and calcium ions. We found that the affinity of mycobacterial transketolase for thiamine diphosphate is by three orders of magnitude lower than that of the human enzyme. Analysis of the structural organization of the active centers of homologous enzymes showed that this difference is due to a replacement of lysine residues by less polar amino acid residues.

Biochemical, Bioinformatic, and Structural Comparisons of Transketolases and Position of Human Transketolase in the Enzyme Evolution

“…A seventh less systemic histidyl residue is also identified (His 281 in the consensus sequence). The literature only describes the role of some of these residues in ec TK and sc TK such as stabilizing the interactions between TK and TPP via pyrophosphate group (His 86, His 284, and His 498 on the consensus TK) and thiazolium ring (His 510). ,,,,,,,,, This crown is also involved in TPP activation by favoring the deprotonation of the thiazolium ring by the N4’ of the aminopyrimidine ring of TPP (His 45, His 121, and His 510) and in activation by Glu 447 (Figure S5). ,, His 45, His 121, and His 510 are close to the conserved Asp 506, forming a subnetwork of four residues and stabilizing the proton released from TPP activation.…”

“…K TPP app values are in the micromolar range, as previously observed for other TKs. This very high affinity between TKs and its cofactor explains why it is often difficult to remove the TPP from the active site as observed for hs TK, mt TK, and pf TK. , Numerous studies have focused on the use of TPP-derived molecules to inhibit TKs (2′-methoxy-thiamine, oxythiamine, 4-anilinoquinoline triazine) in particular in oncology. ,, Taking into account the K TPP app of these TKs, it would be more pertinent to look for inhibitors that bind elsewhere on TKs rather than TPP competitors. , …”

“…We identified a crown of six histidyl residues placed around TPP except for hs TK, which has only five. The role of some of them in the stabilization and activation of TPP has been previously identified in ec TK and sc TK. ,,,,,,,,, Two additional residues involved in TPP activation (Glu) and proton stabilization (Asp) are also conserved. Two of the histidyl residues of the crown are systematically involved in the interaction between the monomers.…”

“…The literature only describes the role of some of these residues in ecTK and scTK such as stabilizing the interactions between TK and TPP via pyrophosphate group (His 86, His 284, and His 498 on the c o n s e n s u s T K ) a n d t h i a z o l i u m r i n g ( H i s 510). 3,14,15,24,54,55,58,59,61,62 This crown is also involved in TPP activation by favoring the deprotonation of the thiazolium 1 plus scTK, Table S8). Logos show residue occurrences for each TK consensus position, with scores determined as described in the experimental section, from dark blue (variable residues) to pink (conserved residues) (Figure S2).…”

Search for Inhibitors of <i>Mycobacterium tuberculosis</i> Transketolase in a Series of Sulfo-Substituted Compounds

“…The recombinant mbTK protein was obtained using the pET-19b plasmid carrying the Rv1449c gene and the Escherichia coli strain BL21(DE3). Protein isolation and purification were performed as described previously [ 8 , 9 ]. The activity of mbTK was measured by the coupled NAD + reduction reaction, catalyzed by glyceraldehyde 3-phosphate dehydrogenase from rabbit muscles [ 10 ].…”