Characterization of M. tuberculosis SerB2, an Essential HAD-Family Phosphatase, Reveals Novel Properties

Abstract

M. tuberculosis harbors an essential phosphoserine phosphatase (MtSerB2, Rv3042c) that contains two small- molecule binding ACT-domains (Pfam 01842) at the N-terminus followed by the phosphoserine phosphatase (PSP) domain. We found that exogenously added MtSerB2 elicits microtubule rearrangements in THP-1 cells. Mutational analysis demonstrates that phosphatase activity is co-related to the elicited rearrangements, while addition of the ACT-domains alone elicits no rearrangements. The enzyme is dimeric, exhibits divalent metal- ion dependency, and is more specific for l- phosphoserine unlike other classical PSPases. Binding of a variety of amino acids to the ACT-domains influences MtSerB2 activity by either acting as activators/inhibitors/have no effects. Additionally, reduced activity of the PSP domain can be enhanced by equimolar addition of the ACT domains. Further, we identified that G18 and G108 of the respective ACT-domains are necessary for ligand-binding and their mutations to G18A and G108A abolish the binding of ligands like l- serine. A specific transition to higher order oligomers is observed upon the addition of l- serine at ∼0.8 molar ratio as supported by Isothermal calorimetry and Size exclusion chromatography experiments. Mutational analysis shows that the transition is dependent on binding of l- serine to the ACT-domains. Furthermore, the higher-order oligomeric form of MtSerB2 is inactive, suggesting that its formation is a mechanism for feedback control of enzyme activity. Inhibition studies involving over eight inhibitors, MtSerB2, and the PSP domain respectively, suggests that targeting the ACT-domains can be an effective strategy for the development of inhibitors.

The M. tuberculosis HAD phosphatase (Rv3042c) interacts with host proteins and is inhibited by Clofazimine

“…MtSerB2 is secreted into culture filtrate It was shown earlier by our group that exogenously added MtSerB2 elicits cytoskeletal rearrangements in the THP-1 cell line 5 . Since mycobacteria induce cytoskeleton rearrangements upon infection [12,13], the subcellular localization and the possibility of secretion of MtSerB2 into the culture filtrate was checked.…”

“…In fact, point mutants of the regulatory ACT domain such as G18A, G108A, and also those of the PSP domain such as D341/ 345N and S273A have either less or no phosphatase activity at all. Further, unlike the wild-type protein, the mutants do not exhibit functional interactions with host phosphoproteins [5]. As part of a long-term inhibitor screening program, we found that Clofazimine inhibits the phosphatase activity of MtSerB2.…”

“…Also, it was found that MtSerB2 induces microtubule rearrangements in THP-1 cells upon exogenous addition of the protein. Additionally, the phosphatase activity of enzyme was found to be co-related to the elicited microtubule rearrangements [5]. Generally, HAD family phosphatases are involved in metabolic processes.…”

“…MtSerB2 contains two ACT domains (small-molecule Electronic supplementary material The online version of this article (doi:10.1007/s00018-016-2177-2) contains supplementary material, which is available to authorized users. binding domains; Pfam 01842) at the N-terminus followed by a classic phosphatase (PSP) domain [5]. The ACT domains of MtSerB2 are largely involved in amino acid binding and modulate the catalytic efficiency of MtSerB2 phosphatase activity.…”

Bacterial Virulence Factors: Secreted for Survival

“…However, since Clorobiocin targets other mycobacterial factors also, the authors suggested a need to develop structural analogs of the identified inhibitors with MtSerB2 specific intracellular activity. Around the same time, Yadav et al [36] published another report on MtSerB2 characterization. MtSerB2 contains two small molecule binding ACT domains (Aspartate kinase, Chorismate mutase, and TyrA protein regulatory domain) at its N-terminus and a classical phosphoserine phosphatase domain (PSP) toward the C-terminal end.…”