van der Peet, P. | Search bioRxiv

bioRxiv2022

Mining the human gut microbiome identifies mycobacterial D-arabinan degrading enzymes

Division and degradation of bacterial cell walls requires coordinated action of a myriad of enzymes. This particularly applies to the elaborate cell walls of acid-fast organisms such as Mycobacterium tuberculosis, which consist of a multi-layered cell wall that contains an unusual glycan called arabinogalactan. Enzymes that cleave the O_SCPLOWDC_SCPLOW-arabinan core of this structure have not previously been identified in any organism. We have interrogated the diverse carbohydrate degrading enzymes expressed by the human gut microbiota and uncovered four families of glycoside hydrolases with the capability to degrade the O_SCPLOWDC_SCPLOW-arabinan or O_SCPLOWDC_SCPLOW-galactan components of arabinogalactan. Using novel exo-O_SCPLOWDC_SCPLOW-galactofuranosidases from gut bacteria we generated enriched O_SCPLOWDC_SCPLOW-arabinan and used it to identify D. gadei as a D-arabinan degrader. This enabled the discovery of endo- and exo-acting enzymes that cleave D-arabinan. We have identified new members of the DUF2961 family (GH172), and a novel family of glycoside hydrolases (DUF4185) that display endo-O_SCPLOWDC_SCPLOW-arabinofuranase activity. The DUF4185 enzymes are conserved in mycobacteria and found in many microbes, suggesting that the ability to degrade mycobacterial glycans plays an important role in the biology of diverse organisms. All mycobacteria encode two conserved endo-O_SCPLOWDC_SCPLOW-arabinanases that display different preferences for the O_SCPLOWDC_SCPLOW-arabinan-containing cell wall components arabinogalactan and lipoarabinomannan, suggesting they are important for cell wall modification and/or degradation. The discovery of these enzymes will support future studies into the structure and function of the mycobacterial cell wall.

biochemistry↗

bioRxiv2018

The metabolic repair enzyme phospho-glycolate phosphatase regulates central carbon metabolism and fosmidomycin sensitivity in Plasmodium falciparum

The asexual blood stages of the malaria parasite, Plasmodium falciparum are highly dependent on glycolysis for ATP synthesis, redox balance and provision of essential anabolic precursors. Recent studies have suggested that members of the haloacid dehalogenase (HAD) family of metabolite phosphatases may play an important role in regulating multiple pathways in P. falciparum central carbon metabolism. Here, we show that the P. falciparum HAD protein, phosphoglycolate phosphatase (PfPGP), which is homologous to yeast Pho13 and mammalian PGP, regulates glycolysis in asexual blood stages by controlling intracellular levels of several intermediates and novel end-products of this pathway. Deletion of the P. falciparum pgp gene significantly attenuated asexual parasite growth in red blood cells, while comprehensive metabolomic analysis revealed the accumulation of two previously uncharacterized metabolites, as well as changes in a number of intermediates in glycolysis and the pentose phosphate pathway. The two unknown metabolites were assigned as 2-phospho-lactate and 4-phosphoerythronate by comparison of their mass spectra with synthetic standards. 2-Phospho-lactate was significantly elevated in wildtype and {Delta}PfPGP parasites cultivated in the presence of methylglyoxal and D-lactate, but not L-lactate, indicating that it is a novel end-product of the methylglyoxal pathway. 4-Phosphoerythronate is a putative side product of the glycolytic enzyme, glyceraldehyde dehydrogenase and the accumulation of both 4-phosphoerythronate and 2-phospho-D-lactate were associated with changes in glycolytic and the pentose phosphate pathway fluxes as shown by 13C-glucose labelling studies and increased sensitivity of the {Delta}PfPGP parasites to the drug fosmidomycin. Our results suggest that PfPGP contributes to a novel futile metabolic cycle involving the phosphorylation/dephosphorylation of D-lactate as well as detoxification of metabolites, such as 4-phosphoerythronate, and both may have important roles in regulating P. falciparum central carbon metabolism.\n\nAuthor summaryThe major pathogenic stages of the malaria parasite, Plasmodium falciparum, develop in red blood cells where they have access to an abundant supply of glucose. Unsurprisingly these parasite stages are addicted to using glucose, which is catabolized in the glycolytic and the pentose phosphate pathways. While these pathways also exist in host cells, there is increasing evidence that P. falciparum has evolved novel ways for regulating glucose metabolism that could be targeted by next-generation of anti-malarial drugs. In this study, we show the red blood cell stages of P. falciparum express an enzyme that is specifically involved in regulating the intracellular levels of two metabolites that are novel end-products or side products of glycolysis. Parasite mutants lacking this enzyme are viable but exhibit diminished growth rates in red blood cells. These mutant lines accumulate the two metabolites, and exhibit global changes in central carbon metabolism. Our findings suggest that metabolic end/side products of glycolysis directly regulate the metabolism of these parasites, and that the intracellular levels of these are tightly controlled by previously uncharacterized metabolite phosphatases.

biochemistry↗