Identification and structural characterization of a histidinol phosphate phosphatase from Mycobacterium tuberculosis

Abstract

The absence of a histidine biosynthesis pathway in humans, coupled with histidine essentiality for survival of the important human pathogen (), underscores the importance of the bacterial enzymes of this pathway as major antituberculosis drug targets. However, the identity of the mycobacterial enzyme that functions as the histidinol phosphate phosphatase (HolPase) of this pathway remains to be established. Here, we demonstrate that the enzyme encoded by the gene, belonging to the inositol monophosphatase (IMPase) family, functions as the HolPase and specifically dephosphorylates histidinol phosphate. The crystal structure of Rv3137 in apo form enabled us to dissect its distinct structural features. Furthermore, the holo-complex structure revealed that a unique cocatalytic multizinc-assisted mode of substrate binding and catalysis is the hallmark of HolPase. Interestingly, the enzyme-substrate complex structure unveiled that although monomers possess individual catalytic sites they share a common product-exit channel at the dimer interface. Furthermore, target-based screening against HolPase identified several small-molecule inhibitors of this enzyme. Taken together, our study unravels the missing enzyme link in the histidine biosynthesis pathway, augments our current mechanistic understanding of histidine production in , and has helped identify potential inhibitors of this bacterial pathway.

De novo histidine biosynthesis protects Mycobacterium tuberculosis from host IFN-γ mediated histidine starvation

“…In order to delineate the role of bacterial histidine biosynthesis in Mtb infection, we examined the dynamics of cellular expression of the histidine biosynthesis pathway enzymes as the infection progresses. For this, three key enzymes of the pathway – imidazole glycerol phosphate dehydratase ( hisB , Rv1601) 24 , histidinol dehydrogenase ( hisD , Rv1599) 18 and histidinol phosphate phosphatase ( hisN , Rv3137) 25 , 26 , well known for their essentiality were chosen. These enzymes were overexpressed and purified to homogeneity using appropriate protocols (Supplementary Fig.…”

“…Expression clones of hisB 10 and hisN 25 were available, whereas hisD expression plasmid was prepared. Briefly, the corresponding ORF was amplified using gene specific primers and cloned into an expression vector pYUB-1062, overexpressed in a M. smegmatis mc 2 -4517 system and the protein was purified to homogeneity by using Ni 2+ -NTA affinity chromatography, followed by size exclusion chromatography.…”

Characterization of a triazole scaffold compound as an inhibitor of Mycobacterium tuberculosis imidazoleglycerol‐phosphate dehydratase

“…This entire complex with both dehydratase and phosphatase activities is commonly known as HisB. However, in other organisms, such as Mtb , 26,27 fungi 28 and plants, 29,30 these enzymes are not fused. They operate separately and are encoded by two separate ORFs.…”

“…Owing to the absence of both structural and functional homologs of IGPD in mammals, this enzyme has been a major anti‐herbs 31‐33 target. IGPD has also been regarded as a promising anti‐bacterial 34,35 and anti‐fungal 36 drug target.Over the past decade, to derive a mechanistic understanding of histidine production by Mtb , which could assist in developing target‐specific anti‐TB small molecules, we have structurally and biochemically characterized IGPD, 26 HisC 37 and HisN 27 of this pathway. IGPD being an important anti‐bacterial drug target, 38 we sought to determine its 3D structure at a higher resolution to obtain a better model comprising more precise structural information for the design of IGPD specific anti‐TB inhibitors through a structure‐guided approach.…”

Serendipitous crystallization and structure determination of bacterioferritin from Achromobacter

“…In order to identify the protein, mass-spectrometric analysis was employed. The protein band of the appropriate size was excised from the SDS polyacrylamide gel and processed as described elsewhere (Jha et al, 2018). The extracted protein was subjected to trypsin digestion, following which the peptides were analyzed by MALDI-TOF MS/MS (TOF/TOF 5800 analyzer, SCIEX, Shanghai, People's Republic of China) for peptide mass fingerprinting.…”