Tamjidmaa Khatanbaatar

• Stocker, Christian; Khatanbaatar, Tamjidmaa; Bressan, Luca; Würth-Roderer, Kathrin; Cordara, Gabriele & Krengel, Ute [Show all 7 contributors for this article] (2023). Novel exported fusion enzymes with chorismate mutase and cyclohexadienyl dehydratase activity: Shikimate pathway enzymes teamed up in no man’s land. Journal of Biological Chemistry. ISSN 0021-9258. 299(10). doi: 10.1016/j.jbc.2023.105161. Full text in Research Archive Show summary

  Chorismate mutase (CM) and cyclohexadienyl dehydratase (CDT) catalyze two subsequent reactions in the intracellular biosynthesis of l-phenylalanine (Phe). Here, we report the discovery of novel and extremely rare bifunctional fusion enzymes, consisting of fused CM and CDT domains, which are exported from the cytoplasm. Such enzymes were found in only nine bacterial species belonging to non-pathogenic γ- or β-Proteobacteria. In γ-proteobacterial fusion enzymes, the CM domain is N-terminal to the CDT domain, whereas the order is inverted in β-Proteobacteria. The CM domains share 15% to 20% sequence identity with the AroQγ class CM holotype of Mycobacterium tuberculosis (∗MtCM), and the CDT domains 40% to 60% identity with the exported monofunctional enzyme of Pseudomonas aeruginosa (PheC). In vitro kinetics revealed a Km <7 μM, much lower than for ∗MtCM, whereas kinetic parameters are similar for CDT domains and PheC. There is no feedback inhibition of CM or CDT by the pathway's end product Phe, and no catalytic benefit of the domain fusion compared with engineered single-domain constructs. The fusion enzymes of Aequoribacter fuscus, Janthinobacterium sp. HH01, and Duganella sacchari were crystallized and their structures refined to 1.6, 1.7, and 2.4 Å resolution, respectively. Neither the crystal structures nor the size-exclusion chromatography show evidence for substrate channeling or higher oligomeric structure that could account for the cooperation of CM and CDT active sites. The genetic neighborhood with genes encoding transporter and substrate binding proteins suggests that these exported bifunctional fusion enzymes may participate in signaling systems rather than in the biosynthesis of Phe.

• Thorbjørnsrud, Helen Vikdal; Bressan, Lucas; Khatanbaatar, Tamjidmaa; Carrer, Manuel; Würth-Roderer, Kathrin & Cordara, Gabriele [Show all 9 contributors for this article] (2023). What Drives Chorismate Mutase to Top Performance? Insights from a Combined In Silico and In Vitro Study. Biochemistry. ISSN 0006-2960. 62(3), p. 782–796. doi: 10.1021/acs.biochem.2c00635. Full text in Research Archive Show summary

  Unlike typical chorismate mutases, the enzyme from Mycobacterium tuberculosis (MtCM) has only low activity on its own. Remarkably, its catalytic efficiency kcat/Km can be boosted more than 100-fold by complex formation with a partner enzyme. Recently, an autonomously fully active MtCM variant was generated using directed evolution, and its structure was solved by X-ray crystallography. However, key residues were involved in crystal contacts, challenging the functional interpretation of the structural changes. Here, we address these challenges by microsecond molecular dynamics simulations, followed up by additional kinetic and structural analyses of selected sets of specifically engineered enzyme variants. A comparison of wild-type MtCM with naturally and artificially activated MtCMs revealed the overall dynamic profiles of these enzymes as well as key interactions between the C-terminus and the active site loop. In the artificially evolved variant of this model enzyme, this loop is preorganized and stabilized by Pro52 and Asp55, two highly conserved residues in typical, highly active chorismate mutases. Asp55 stretches across the active site and helps to appropriately position active site residues Arg18 and Arg46 for catalysis. The role of Asp55 can be taken over by another acidic residue, if introduced at position 88 close to the C-terminus of MtCM, as suggested by molecular dynamics simulations and confirmed by kinetic investigations of engineered variants.

• Thorbjørnsrud, Helen Vikdal; Bressan, Luca; Khatanbaatar, Tamjidmaa; Carrer, Manuel; Würth-Roderer, Kathrin & Cordara, Gabriele [Show all 9 contributors for this article] (2022). What drives chorismate mutase to top performance? Insights from a combined in silico and in vitro study. BioRxiv. ISSN 0362-4331. doi: 10.1101/2022.11.08.515563. Full text in Research Archive

View all works in NVA

• Stocker, Christian; Khatanbaatar, Tamjidmaa; Bressan, Luca; Würth-Roderer, Kathrin; Cordara, Gabriele & Krengel, Ute [Show all 7 contributors for this article] (2023). Novel exported bifunctional fusion enzymes with chorismate mutase and cyclohexadienyl dehydratase activity: shikimate pathway enzymes teamed up in no man's land. BioRxiv. ISSN 0362-4331. doi: 10.1101/2023.03.13.532365. Full text in Research Archive
• Khatanbaatar, Tamjidmaa (2022). Structure-function relationship of metabolic fusion enzymes from the shikimate pathway. Full text in Research Archive
• Khatanbaatar, Tamjidmaa (2022). Structure-function analysis of bifunctional metabolic enzymes from the shikimate pathway. Full text in Research Archive
• Khatanbaatar, Tamjidmaa & Krengel, Ute (2023). Partners in crime: Structure-function studies on chorismate mutases and their associated enzymes. Universitetet i Oslo. Full text in Research Archive Show summary

  Aromatic amino acids are essential building blocks for proteins. Humans lack enzymes that produce aromatic amino acids, thus they must consume them with their diet. In contrast, plants, bacteria and fungi are capable of de novo synthesis of aromatic amino acids, including the essential amino acids phenylalanine and tryptophan. Enzymes involved in the biosynthesis of aromatic amino acids are very promising drug targets, to fight bacterial and fungal infections or plant parasitosis. This PhD thesis is concerned with the study of chorismate mutase (CM), a fundamental metabolic enzyme. CM is often found associated to other enzymes from the same biosynthetic pathway, which regulate its activity. This work reports the structure of chorismate mutases from several bacteria, including human pathogens Mycobacterium tuberculosis and Pseudomonas aeruginosa, and novel bifunctional chorismate mutases from non-pathogenic bacteria. We shed light on their activity and regulation, including the mode of interaction with associated enzymes, laying the foundation for the discovery of new drugs.

View all works in NVA