Blood Group Antigen Recognition by a Solute-Binding Protein from a Serotype 3 Strain of Streptococcus pneumoniae

Streptococcus pneumoniae is a common bacterial pathogen that is well known for its ability to cause acute respiratory disease (pneumonia), ear infections, and other serious illnesses. This Gram-positive bacterium relies on its carbohydrate-metabolizing capabilities for full virulence in its host; however, the range of glycan targets that it can attack is presently not fully appreciated. S. pneumoniae is known to have a fucose utilization operon that in the TIGR4 strain plays a role in its virulence. Here we identify a second type of fucose utilization operon that is present in a subset of S. pneumoniae strains, including the serotype 3 strain SP3-BS71. This operon contains a transporter with a solute-binding protein, FcsSBP (fucose solute-binding protein), that interacts tightly (K_a ∼ 1 × 10⁶ M^− 1) and specifically with soluble A- and B-antigen trisaccharides but displays no selectivity between these two sugars. The structure of the FcsSBP in complex with the A-trisaccharide antigen, determined to 2.35 Å, reveals its mode of binding to the reducing end of this sugar, thus highlighting this protein's requirement for soluble blood group antigen ligands. Overall, this report exposes a heretofore unknown capability of certain S. pneumoniae strains to transport and potentially metabolize the histo-blood group antigen carbohydrates of its host.     

Constitutive activation of the fucAO operon and silencing of the divergently transcribed fucPIK operon by an IS5 element in Escherichia coli mutants selected for growth on L-1,2-propanediol.

L-1,2-Propanediol is an irretrievable end product of L-fucose fermentation by Escherichia coli. Selection for increased aerobic growth rate on propanediol results in the escalation of basal synthesis of the NAD+-linked oxidoreductase encoded by fucO, a member of the fuc regulon for the utilization of L-fucose. In general, when fucO becomes constitutively expressed, two other simultaneous changes occur: the fucA gene encoding fuculose-1-phosphate aldolase becomes constitutively expressed and the fucPIK operon encoding fucose permease, fucose isomerase, and fuculose kinase becomes noninducible. In the present study, we show that fucO and fucA form an operon which is divergently transcribed from the adjacent fucPIK operon. In propanediol-positive and fucose-negative mutants the cis-controlling region shared by the operons fucAO and fucPIK is lengthened by 1.2 kilobases. DNA hybridization identified the insertion element to be IS5. This element, always oriented in the same direction with the left end (the BglII end) proximal to fucA, apparently causes constitutive expression of fucAO and noninducibility of fucPIK. The DNA of the fucAO operon and a part of the adjacent fucP was sequenced.     

Differential Recognition and Hydrolysis of Host Carbohydrate Antigens by Streptococcus pneumoniae Family 98 Glycoside Hydrolases

The presence of a fucose utilization operon in the Streptococcus pneumoniae genome and its established importance in virulence indicates a reliance of this bacterium on the harvesting of host fucose-containing glycans. The identities of these glycans, however, and how they are harvested is presently unknown. The biochemical and high resolution x-ray crystallographic analysis of two family 98 glycoside hydrolases (GH98s) from distinctive forms of the fucose utilization operon that originate from different S. pneumoniae strains reveal that one enzyme, the predominant type among pneumococcal isolates, has a unique endo-β-galactosidase activity on the Lewis^Y antigen. Altered active site topography in the other species of GH98 enzyme tune its endo-β-galactosidase activity to the blood group A and B antigens. Despite their different specificities, these enzymes, and by extension all family 98 glycoside hydrolases, use an inverting catalytic mechanism. Many bacterial and viral pathogens exploit host carbohydrate antigens for adherence as a precursor to colonization or infection. However, this is the first evidence of bacterial endoglycosidase enzymes that are known to play a role in virulence and are specific for distinct host carbohydrate antigens. The strain-specific distribution of two distinct types of GH98 enzymes further suggests that S. pneumoniae strains may specialize to exploit host-specific antigens that vary from host to host, a factor that may feature in whether a strain is capable of colonizing a host or establishing an invasive infection.Streptococcus pneumoniae asymptomatically colonizes the nasopharynx of 10–40% of people, but given the appropriate opportunity, it can become an extremely aggressive pathogen (1–3). This bacterium causes millions of deaths annually (1), is acquiring antibiotic resistance (4), and shows a disturbing and lethal synergy with the Influenza virus (5). The ability of S. pneumoniae to cause invasive disease is increasingly being linked with the capacity of this bacterium to attack and process the glycans present in host tissues (see Ref. 6 for a review). Indeed, large scale screening of pneumococcal virulence factors has revealed a large complement of genes devoted to complex carbohydrate metabolism that contribute to pneumococcal virulence (7–9). Recent elegant studies have focused on showing how a group of three exo-glycosidases sequentially trim complex human N-glycans (10, 11). These enzymes, however, only make up a fraction of the 39 glycosidases predicted to be in the pneumococcal genome (TIGR4 strain); at least 18 of these 39 are required for full virulence of the bacterium (7). Despite the growing appreciation for the role of carbohydrate metabolism in pneumococcal virulence and the possibility of targeting such metabolic pathways with small molecule therapeutic compounds, the bulk of the carbohydrate-active proteins of S. pneumoniae remain unexamined. As such, we presently have a relatively superficial but growing appreciation for the array of host glycans that S. pneumoniae can degrade.Several S. pneumoniae genes whose protein products are dedicated to the harvesting and processing of the sugar fucose are beginning to emerge as an important set of pneumococcal virulence factors (12). Comparative genomic studies of several S. pneumoniae genomes has suggested genetic variability at this locus; however, some components of the operon were observed to be present in all of the studied isolates (13). Through our recent identification and characterization of a novel solute-binding protein present in an alternate pneumococcal fucose utilization operon, we have made the observation that there are two different fucose utilization operons distributed among pneumococcal strains (14). Although the organization and composition of the two operons is different, both pathways are predicted to be initiated by the action of a family 98 glycoside hydrolase that is probably secreted (for a discussion of the sequence classification system of glycoside hydrolases, see Ref. 15). This GH98 is the same as that identified as a virulence factor in the TIGR4 strain (7). Remarkably, the GH98 enzymes from the two different pathways display different modular architectures, and their shared catalytic modules only have modest amino acid sequence identity. Given the placement of these enzymes in a fucose utilization operon, we hypothesized that they have activity on fucose-containing glycans; however, their divergent sequences and different modular arrangements led us to postulate that they would have different glycan substrate specificities.Here we describe the specificity and catalytic mechanism for these two different types of S. pneumoniae GH98 enzymes, one from the TIGR4 strain (Sp4GH98) and the other from the SP3-BS71 strain (Sp3GH98). Both enzymes act as endo-β-1,4-galactosidases on the galactosyl-β-1,4-N-acetylglucosamine linkage found in type 2 carbohydrate blood group antigens, although Sp4GH98 displays specificity for the Lewis^Y antigen, whereas Sp3GH98 is highly selective for the same linkage in the blood group A/B-antigens. The biochemical analysis of these enzymes in combination with the determination of their structures in complex with products and substrates provides molecular level insight to their catalytic mechanism and how they discriminate between their respective substrates. We discuss these results in the context of the recent association of the pneumococcal fucose utilization operon with the virulence of S. pneumoniae (7, 12) and the possible strain-specific dependence of pneumococcal virulence on the carbohydrate antigens presented by different hosts.     

Isolation of a sn-glycerol 3-phosphate: NAD oxidoreductase from spinach leaves.

An NAD-dependent glycerol 3-phosphate dehydrogenase (sn-glycerol 3-phosphate: NAD oxidoreductase; EC 1.1.1.8) has been purified from spinach leaves by a three-step procedure involving ion-exchange, gel filtration, and affinity chromatography. The enzyme has been purified over 10,000-fold to a specific activity of 38. It has a molecular weight of approximately 63,500. The pH optimum for the reduction of dihydroxyacetone phosphate is 6.8 and for glycerol 3-phosphate oxidation it is 9.5. During dihydroxyacetone phosphate reduction hyperbolic kinetics were observed when either NADH or dihydroxyacetone phosphate was the variable substrate, but concentrations of NADH greater than 150 μm were inhibitory. Michaelis constants were 0.30–0.35 mm for dihydroxyacetone phosphate and 0.01 mm for NADH. Glycerol 3-phosphate oxidation obeyed Michaelis-Menten kinetics with a K_m of 0.19 mm for NAD and 1.6 mm for glycerol 3-phosphate. The enzyme was specific for those substrates, and dihydroxyacetone, glyceraldehyde, glyceraldehyde 3-phosphate, NADPH, NADP, and glycerol were not utilized. The spinach leaf enzyme appears to be in the cytoplasm and probably functions for the production of glycerol 3-phosphate from dihydroxyacetone phosphate.     

A mutant crp allele that differentially activates the operons of the fuc regulon in Escherichia coli.

L-Fucose is used by Escherichia coli through an inducible pathway mediated by a fucP-encoded permease, a fucI-encoded isomerase, a fucK-encoded kinase, and a fucA-encoded aldolase. The adolase catalyzes the formation of dihydroxyacetone phosphate and L-lactaldehyde. Anaerobically, lactaldehyde is converted by a fucO-encoded oxidoreductase to L-1,2-propanediol, which is excreted. The fuc genes belong to a regulon comprising four linked operons: fucO, fucA, fucPIK, and fucR. The positive regulator encoded by fucR responds to fuculose 1-phosphate as the effector. Mutants serially selected for aerobic growth on propanediol became constitutive in fucO and fucA [fucO(Con) fucA(Con)], but noninducible in fucPIK [fucPIK(Non)]. An external suppressor mutation that restored growth on fucose caused constitutive expression of fucPIK. Results from this study indicate that this suppressor mutation occurred in crp, which encodes the cyclic AMP-binding (or receptor) protein. When the suppressor allele (crp-201) was transduced into wild-type strains, the recipient became fucose negative and fucose sensitive (with glycerol as the carbon and energy source) because of impaired expression of fucA. The fucPIK operon became hyperinducible. The growth rate on maltose was significantly reduced, but growth on L-rhamnose, D-galactose, L-arabinose, glycerol, or glycerol 3-phosphate was close to normal. Lysogenization of fuc+ crp-201 cells by a lambda bacteriophage bearing crp+ restored normal growth ability on fucose. In contrast, lysogenization of [fucO(Con)fucA(Con)fucPIK(Non)crp-201] cells by the same phage retarded their growth on fucose.     

Biochemical characterisation of triose phosphate isomerase from the liver fluke Fasciola hepatica

Triose phosphate isomerase (TPI) catalyses the interconversion of dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, a reaction in the glycolytic pathway. TPI from the common liver fluke, Fasciola hepatica, has been cloned, sequenced and recombinantly expressed in Escherichia coli. The protein has a monomeric molecular mass of approximately 28 kDa. Crosslinking and gel filtration experiments demonstrated that the enzyme exists predominantly as a dimer in solution. F. hepatica TPI is predicted to have a β-barrel structure and key active site residues (Lys-14, His-95 and Glu-165) are conserved. The enzyme shows remarkable stability to both proteolytic degradation and thermal denaturation. The melting temperature, estimated by thermal scanning fluorimetry, was 67 °C and this temperature was increased in the presence of either dihydroxyacetone phosphate or glyceraldehyde 3-phosphate. Kinetic studies showed that F. hepatica TPI demonstrates Michaelis–Menten kinetics in both directions, with K_m values for dihydroxyacetone phosphate and glyceraldehyde 3-phosphate of 2.3 mM and 0.66 mM respectively. Turnover numbers were estimated at 25,000 s⁻¹ for the conversion of dihydroxyacetone phosphate and 1900 s⁻¹ for the conversion of glyceraldehyde 3-phosphate. Phosphoenolpyruvate acts as a weak inhibitor of the enzyme. F. hepatica TPI has many features in common with mammalian TPI enzymes (e.g. β-barrel structure, homodimeric nature, high stability and rapid kinetic turnover). Nevertheless, recent successful identification of specific inhibitors of TPI from other parasites, suggests that small differences in structure and biochemical properties could be exploited in the development of novel, species-specific inhibitors.     

Characterization of aldolase from Methanococcus jannaschii by gas chromatography

The products of reactions catalyzed by Methanococcus. jannaschii (Mj) aldolase using various substrates were identified by gas chromatography (GC). Although Mj aldolase is considered a fuculose-1-phosphate aldolase based on homology searching after gene sequencing, it has not been proven to be a fuculose-1-phosphate aldolase based on its reaction products. Mj aldolase was found to catalyze reactions between glycoaldehyde or D, L-glyceraldehyde and DHAP (dihydroxyacetone phosphate). Before performing GC the ketoses produced were converted into peracetylated alditol derivatives by sequential reactions, i.e., dephosphorylation, NaBH(4) reduction, and acetylation. By comparing the GC data of final products with those of standard alditol samples, it was found that the enzymatic reactions with glycoaldehyde, D-glyceraldehyde, and D, L-glyceraldehyde produced D-ribulose-1-phosphate, D-psicose-1-phosphate, and a mixture of D-psicose and L-tagatose-1-phosphate, respectively. These results provide direct evidence that Mj aldolase is a fuculose-1-phosphate aldolase.     

Dual Mechanisms of Metabolite Acquisition by the Obligate Intracytosolic Pathogen Rickettsia prowazekii Reveal Novel Aspects of Triose Phosphate Transport

Rickettsia prowazekii is an obligate intracytosolic pathogen and the causative agent of epidemic typhus fever in humans. As an evolutionary model of intracellular pathogenesis, rickettsiae are notorious for their use of transport systems that parasitize eukaryotic host cell biochemical pathways. Rickettsial transport systems for substrates found only in eukaryotic cell cytoplasm are uncommon among free-living microorganisms and often possess distinctive mechanisms. We previously reported that R. prowazekii acquires triose phosphates for phospholipid biosynthesis via the coordinated activities of a novel dihydroxyacetone phosphate transport system and an sn-glycerol-3-phosphate dehydrogenase (K. M. Frohlich et al., J. Bacteriol. 192:4281–4288, 2010). In the present study, we have determined that R. prowazekii utilizes a second, independent triose phosphate acquisition pathway whereby sn-glycerol-3-phosphate is directly transported and incorporated into phospholipids. Herein we describe the sn-glycerol-3-phosphate and dihydroxyacetone phosphate transport systems in isolated R. prowazekii with respect to kinetics, energy coupling, transport mechanisms, and substrate specificity. These data suggest the existence of multiple rickettsial triose phosphate transport systems. Furthermore, the R. prowazekii dihydroxyacetone phosphate transport systems displayed unexpected mechanistic properties compared to well-characterized triose phosphate transport systems from plant plastids. Questions regarding possible roles for dual-substrate acquisition pathways as metabolic virulence factors in the context of a pathogen undergoing reductive evolution are discussed.     

Structural characterization of the PTS IIA and IIB proteins associated with pneumococcal fucose utilization

Streptococcus pneumoniae harbors a significant number of transporters, including phosphotransferase (PTS) systems, allowing the bacterium to utilize a number of different carbohydrates for metabolic and other purposes. The genes encoding for one PTS transport system in particular (EII^fuc) are found within a fucose utilization operon in S. pneumoniae TIGR4. Here, we report the three‐dimensional structures of IIA^fuc and IIB^fuc providing evidence that this PTS system belongs to the EII^man family. Additionally, the predicted metabolic pathway for this distinctive fucose utilization system suggests that EII^fuc transports the H‐disaccharide blood group antigen, which would represent a novel PTS transporter specificity. Proteins 2017; 85:963–968. © 2016 Wiley Periodicals, Inc.     

Lactose and D0galactose metabolism in Staphylococcus aureus: pathway of D-galactose 6-phosphate degradation

The pathway by which D-galactose 6-phosphate is degraded in Staphylococcus aureus has been elucidated. Galactose 6-phosphate is isomerized to tagatose 6-phosphate, which is phosphorylated with adenosine 5′-triphosphate, and the resulting tagatose 1,6-diphosphate is cleaved to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. The isomerase, kinase, and aldolase that catalyze these reactions are inducible and are distinct from the corresponding enzymes of glucose 6-phosphate metabolism.     

A metabolic bypass of the triosephosphate isomerase reaction

Triosephosphate isomerase (TIM) catalyzes the interconversion of d-glyceraldehyde 3-phosphate and dihydroxyacetone phosphate, an essential step in glycolytic and gluconeogenic metabolism. To uncover promiscuous isomerases embedded within the Escherichia coli genome, we searched for genes capable of restoring growth of a TIM-deficient bacterium under gluconeogenic conditions. Rather than discovering an isomerase, we selected yghZ, a gene encoding a member of the aldo-keto reductase superfamily. Here we show that YghZ catalyzes the stereospecific, NADPH-dependent reduction of l-glyceraldehyde 3-phosphate, the enantiomer of the TIM substrate. This transformation provides an alternate pathway to the formation of dihydroxyacetone phosphate.     

Characterization of a novel fucose-regulated promoter (PfcsK) suitable for gene essentiality and antibacterial mode-of-action studies in Streptococcus pneumoniae

The promoter of the Streptococcus pneumoniae putative fuculose kinase gene (fcsK), the first gene of a novel fucose utilization operon, is induced by fucose and repressed by glucose or sucrose. When the streptococcal polypeptide deformylase (PDF) gene (def1, encoding PDF) was placed under the control of P(fcsK), fucose-dependent growth of the S. pneumoniae (P(fcsK)::def1) strain was observed, confirming the essential nature of PDF in this organism. The mode of antibacterial action of actinonin, a known PDF inhibitor, was also confirmed with this strain. The endogenous fuculose kinase promoter is a tightly regulated, titratable promoter which will be useful for target validation and for confirmation of the mode of action of novel antibacterial drugs in S. pneumoniae.     

Characterization of Streptococcus pneumoniae N-acetylglucosamine-6-phosphate deacetylase as a novel diagnostic marker

The identification of novel diagnostic markers of pathogenic bacteria is essential for improving the accuracy of diagnoses and for developing targeted vaccines. Streptococcus pneumoniae is a significant human pathogenic bacterium that causes pneumonia. N-acetylglucosamine-6-phosphate deacetylase (NagA) was identified in a protein mixture secreted by S. pneumoniae and its strong immunogenicity was confirmed in an immuno-proteomic assay against the anti-serum of the secreted protein mixture. In this study, recombinant S. pneumoniae NagA protein was expressed and purified to analyze its protein characteristics, immunospecificity, and immunogenicity, thereby facilitating its evaluation as a novel diagnostic marker for S. pneumoniae. Mass spectrometry analysis showed that S. pneumoniae NagA contains four internal disulfide bonds and that it does not undergo post-translational modification. S. pneumoniae NagA antibodies successfully detected NagA from different S. pneumoniae strains, whereas NagA from other pathogenic bacteria species was not detected. In addition, mice infected with S. pneumoniae generated NagA antibodies in an effective manner. These results suggest that NagA has potential as a novel diagnostic marker for S. pneumoniae because of its high immunogenicity and immunospecificity.     

Inhibition of Nicotinamide Phosphoribosyltransferase (NAMPT), an Enzyme Essential for NAD+ Biosynthesis,Leads to Altered Carbohydrate Metabolism in Cancer Cells

Nicotinamide phosphoribosyltransferase (NAMPT) has been extensively studied due to its essential role in NAD⁺ biosynthesis in cancer cells and the prospect of developing novel therapeutics. To understand how NAMPT regulates cellular metabolism, we have shown that the treatment with FK866, a specific NAMPT inhibitor, leads to attenuation of glycolysis by blocking the glyceraldehyde 3-phosphate dehydrogenase step (Tan, B., Young, D. A., Lu, Z. H., Wang, T., Meier, T. I., Shepard, R. L., Roth, K., Zhai, Y., Huss, K., Kuo, M. S., Gillig, J., Parthasarathy, S., Burkholder, T. P., Smith, M. C., Geeganage, S., and Zhao, G. (2013) Pharmacological inhibition of nicotinamide phosphoribosyltransferase (NAMPT), an enzyme essential for NAD⁺ biosynthesis, in human cancer cells: metabolic basis and potential clinical implications. J. Biol. Chem. 288, 3500–3511). Due to technical limitations, we failed to separate isotopomers of phosphorylated sugars. In this study, we developed an enabling LC-MS methodology. Using this, we confirmed the previous findings and also showed that NAMPT inhibition led to accumulation of fructose 1-phosphate and sedoheptulose 1-phosphate but not glucose 6-phosphate, fructose 6-phosphate, and sedoheptulose 7-phosphate as previously thought. To investigate the metabolic basis of the metabolite formation, we carried out biochemical and cellular studies and established the following. First, glucose-labeling studies indicated that fructose 1-phosphate was derived from dihydroxyacetone phosphate and glyceraldehyde, and sedoheptulose 1-phosphate was derived from dihydroxyacetone phosphate and erythrose via an aldolase reaction. Second, biochemical studies showed that aldolase indeed catalyzed these reactions. Third, glyceraldehyde- and erythrose-labeling studies showed increased incorporation of corresponding labels into fructose 1-phosphate and sedoheptulose 1-phosphate in FK866-treated cells. Fourth, NAMPT inhibition led to increased glyceraldehyde and erythrose levels in the cell. Finally, glucose-labeling studies showed accumulated fructose 1,6-bisphosphate in FK866-treated cells mainly derived from dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. Taken together, this study shows that NAMPT inhibition leads to attenuation of glycolysis, resulting in further perturbation of carbohydrate metabolism in cancer cells. The potential clinical implications of these findings are also discussed.     

Transaldolase: From biochemistry to human disease

The role of the enzyme transaldolase (TAL) in central metabolism, its biochemical properties, structure, and role in human disease is reviewed. The nearly ubiquitous enzyme transaldolase is a part of the pentose phosphate pathway and transfers a dihydroxyacetone group from donor compounds (fructose 6-phosphate or sedoheptulose 7-phosphate) to aldehyde acceptor compounds. The phylogeny of transaldolases shows that five subfamilies can be distinguished, three of them with proven TAL enzyme activity, one with unclear function, and the fifth subfamily comprises transaldolase-related enzymes, the recently discovered fructose 6-phosphate aldolases. The three-dimensional structure of a bacterial (Escherichia coli TAL B) and the human enzyme (TALDO1) has been solved. Based on the 3D-structure and mutagenesis studies, the reaction mechanism was deduced. The cofactor-less enzyme proceeds with a Schiff base intermediate (bound dihydroxyacetone). While a transaldolase deficiency is well tolerated in many microorganisms, it leads to severe symptoms in homozygous TAL-deficient human patients. The involvement of TAL in oxidative stress and apoptosis, in multiple sclerosis, and in cancer is discussed.     

Comparative studies on glycerol 3-phosphate dehydrogenase in bees and wasps

Electrophoresis of tissue extracts has shown the presence of multiple electrophoretic forms of glycerol 3-phosphate dehydrogenase (EC 1.1.1.8) in many Hymenoptera. The patterns are most complex in the two bumblebee genera, Bombus and Psithyrus, where from five to six variants are observed.Homogeneous preparations of the major flight muscle variant of glycerol 3-phosphate dehydrogenase have been isolated from thoraces of three bumblebee species and of yellow jacket. The amino acid compositions of these four enzymes plus that from the honeybee have been determined and compared.The Michaelis constant for dihydroxyacetone phosphate was measured for the honeybee, bumblebee, and yellow jacket enzymes. Differences were observed between species but not between bumblebee isozymes.B. nevadensis glycerol 3-phosphate dehydrogenase binds reversibly to both B. nevadensis and honeybee actin. The alar-muscular variants bind more strongly than the omniregional variants.     

Streptococcus pneumoniae secretes a glyceraldehyde-3-phosphate dehydrogenase,which binds haemoglobin and haem

Streptococcus pneumoniae is a gram positive encapsulated bacterium responsible of septicaemia and upper respiratory infections in children. This pathogen requires iron to survive in the host, which it can obtain of haemoglobin (Hb) or haem. Only two Hb-binding membrane proteins have been identified up to now. However it is unknown whether this pathogen secretes proteins in order to scavenge iron from the Hb or haem. Therefore, in order to explore these possibilities, cellular growth of S. pneumoniae was tested with several alternative iron supplies. The bacterial growth was supported with iron, Hb and haem. Additionally, S. pneumoniae expressed and secreted a protein of 38 kDa which was purified and characterized as Hb and haem-binding protein. This protein was also identified by mass spectrometry as glyceraldehyde-3-phosphate dehydrogenase. Our overall results suggest that S. pneumoniae secretes a protein capable of binding two usefull iron sources for this bacterium (Hb and haem). This protein could be playing a dynamic role in the success of the invasive and infective processes of this pathogen.     

Phosphonomethyl analogues of phosphate ester glycolytic intermediates

Analogues of dihydroxyacetone phosphate and of 3-phosphoglycerate were made in which the phosphate group, –O–PO₃H₂, is replaced by the phosphonomethyl group, –CH₂–PO₃H₂. The analogue of dihydroxyacetone phosphate is a substrate for aldolase and glycerol 1-phosphate dehydrogenase (Stribling, 1974), but not for triose phosphate isomerase. The analogue of 3-phosphoglycerate oxidizes NADH under the combined action of 3-phosphoglycerate kinase and glyceraldehyde 3-phosphate dehydrogenase if ATP is added. Thus four out of the five glycolytic enzymes tested handle the phosphonomethyl compounds like the natural phosphates.