Tagatose 6-phosphate Research Articles

Isomer selectivity of one- and two-dimensional approaches of mixed-mode and hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry for sugar phosphates of glycolysis and pentose phosphate pathways

In this study, the chromatographic behavior of mixed-mode and hydrophilic interaction liquid chromatography (HILIC) with the mixed-mode HILIC/strong anion-exchange (SAX) column HILICpak VT-50 2D and the two HILIC columns Atlantis Premier BEH Z-HILIC and Acquity Premier BEH Amide was assessed with regard to their separation capability of the metabolites from the glycolysis and pentose phosphate pathways. Chromatographic conditions were evaluated with the aim of achieving separation of the isomeric glycolytic phosphorylated carbohydrate metabolites free from isomeric interferences and thus allowing for selective targeted analysis by liquid chromatography with tandem mass spectrometry (MS/MS) using multiple reaction monitoring acquisition. The effects of pH values (8.0/9.0/10.0) of the ammonium bicarbonate buffer and gradient time were investigated during HILIC-MS/MS analysis, with the optimal conditions found at pH = 10.0. Separation of the pentose phosphate isomers (ribose 5- and 1-phosphate, xylulose 5-phosphate and ribulose 5-phosphate) was achieved on the mixed-mode HILIC/SAX (HILICpak VT-50 2D) column and HILIC BEH Amide column. Column performance was evaluated based on the direct comparison of chromatographic parameters, i.e. peak width at 50% and peak tailing factors of the individual metabolites. Parity plots were generated allowing a direct comparison between the normalized retention times and assessment of orthogonality of all 3 stationary phases evaluated. Separation of 7 biologically relevant hexose monophosphates metabolites turned out to be challenging by HILIC-MS/MS, with the BEH Amide providing the best individual results for such a separation. However, fructose 6-phosphate and glucose 1-phosphate co-eluted. Therefore, an on-line heart-cutting HILIC-Mixed Mode 2D-LC-QToF experiment was conducted, allowing the separation of this critical isomer pair. In this setup, the BEH Amide column in the 1D separated the majority of target metabolites, while a heart-cut of the peak from totally coeluted fructose 6-phosphate and glucose 1-phosphate was separated in the 2D with HILICpak VT50-2D column, thus allowing undisturbed determination of the glycolytic phosphorylated carbohydrate metabolites due to their chromatographic separation from hexose monophosphate metabolites. The assay specificity towards 7 common hexose monophosphates was characterized (glucose 1- and 6-phosphate, galactose 1- and 6-phosphate, fructose 6-phosphate, mannose 1- and 6-phosphate). The selectivity of some rare hexose monophosphates (allose 6-phosphate, tagatose 6-phosphate, sorbose 1-phosphate) was also tested.

Effect of carbon catabolite repression on lactose and galactose catabolism in Lacticaseibacillus paracasei

Previous research has identified phosphoenolpyruvate-dependent phosphotransferase system and tagatose-6-phosphate (T6P) pathway of lactose/galactose metabolism in Lacticaseibacillus casei groups, which led to no galactose accumulation in fermented milk produced by Lb. paracasei. However, Lb. paracasei preferred lactose to galactose when grown in a mixture of the two sugars, indicating possibility of carbon catabolite repression of galactose catabolism in the fermented milk. Therefore, galactose accumulation was still observed during co-culturing of Lb. paracasei with Streptococcus thermophilus in fermented milk. We demonstrated that the transcriptional regulator lacR acts as a transcriptional repressor of the T6P pathway genes (lacA, lacC and lacD) and lacT acts as a transcriptional activator for genes (lacE) encoding the lactose-transporting PTS in the mixed lactose/galactose CDM medium, indicating possible dual repressive effects of global or local regulation on the carbon catabolism of galactose in Lb. paracasei.

Site-Specific Mutations of GalR Affect Galactose Metabolism in Streptococcus pneumoniae.

Streptococcus pneumoniae (the pneumococcus) is a formidable human pathogen that is capable of asymptomatically colonizing the nasopharynx. Progression from colonization to invasive disease involves adaptation to distinct host niches, which vary markedly in the availability of key nutrients such as sugars. We previously reported that cell-cell signaling via the autoinducer 2 (AI-2)/LuxS quorum-sensing system boosts the capacity of S. pneumoniae to utilize galactose as a carbon source by upregulation of the Leloir pathway. This resulted in increased capsular polysaccharide production and a hypervirulent phenotype. We hypothesized that this effect was mediated by phosphorylation of GalR, the transcriptional activator of the Leloir pathway. GalR is known to possess three putative phosphorylation sites, S317, T319, and T323. In the present study, derivatives of S. pneumoniae D39 with putative phosphorylation-blocking alanine substitution mutations at each of these GalR sites (singly or in combination) were constructed. Growth assays and transcriptional analyses revealed complex phenotypes for these GalR mutants, with impacts on the regulation of both the Leloir and tagatose 6-phosphate pathways. The alanine substitution mutations significantly reduced the capacity of pneumococci to colonize the nasopharynx, middle ear, and lungs in a murine intranasal challenge model.IMPORTANCE Pneumococcal survival in the host and capacity to transition from a commensal to a pathogenic lifestyle are closely linked to the organism's ability to utilize specific nutrients in distinct niches. Galactose is a major carbon source for pneumococci in the upper respiratory tract. We have shown that both the Leloir and tagatose 6-phosphate pathways are necessary for pneumococcal growth in galactose and demonstrated GalR-mediated interplay between the two pathways. Moreover, the three putative phosphorylation sites in the transcriptional regulator GalR play a critical role in galactose metabolism and are important for pneumococcal colonization of the nasopharynx, middle ear, and lungs.

Efficient biocatalytic synthesis of D-tagatose 1,6-diphosphate by LacC-catalysed phosphorylation of D-tagatose 6-phosphate

D-tagatose 1,6-diphosphate is an important metabolite which can be formed via the classical tagatose 6-phosphate pathway or via the tagatose 1-phosphate pathway discovered more recently. The chiral metabolite naturally occurs as an intermediate in the galactose metabolism of various organisms, where it is formed by a reversible condensation of dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphate, whereas D-tagatose 6-phosphate has been synthesized chemically, no chemical synthetic method for further phosphorylation to the D-tagatose 1,6-diphosphate has been described so far. Therefore, a site-specific biocatalytic phosphorylation of the 1-position in D-tagatose 6-phosphate has been chosen. The lacC gene from Lactococcus lactis subsp. lactis was synthesized, cloned into an appropriate expression vector to allow for the production of LacC, the D-tagatose 6-phosphate kinase in Escherichia coli. Subsequently, an efficient biocatalytic synthesis has been developed for the phosphorylation of D-tagatose 6-phosphate at its C1-position by using the recombinant D-tagatose 6-phosphate kinase and the phosphoenolpyruvate/pyruvate kinase-system for ATP regeneration. This straightforward and scalable one-step biocatalytic synthesis of D-tagatose 1,6-diphosphate was successfully scaled up to the gram scale.

The potential of species-specific tagatose-6-phosphate (T6P) pathway in Lactobacillus casei group for galactose reduction in fermented dairy foods

Residual lactose and galactose in fermented dairy foods leads to several industrial and health concerns. There is very little information pertaining to manufacture of fermented dairy foods that are low in lactose and galactose. In the present study, comparative genomic survey demonstrated the constant presence of chromosome-encoded tagatose-6-phosphate (T6P) pathway in Lactobacillus casei group. Lactose/galactose utilization tests and β-galactosidase assay suggest that PTSGal system, PTSLac system and T6P pathway are major contributors for lactose/galactose catabolism in this group of organisms. In addition, it was found than lactose catabolism by Lb. casei group accumulated very limited galactose in the MRS-lactose medium and in reconstituted skim milk, whereas Streptococcus thermophilus and Lb. delbrueckii subsp. bulgaricus (Lb. bulgaricus) strains secreted high amount of galactose extracellularly. Moreover, co-culturing Lb. casei group with Str. thermophilus showed significant reduction in galactose content, while co-culturing Lb. casei group with Lb. bulgaricus showed significant reduction in lactose content but significant increase in galactose content in milk. Overall, the present study highlighted the potential of Lb. casei group for reducing galactose accumulation in fermented milks due to its species-specific T6P pathway.

Novel mode of inhibition by D-tagatose 6-phosphate through a Heyns rearrangement in the active site of transaldolase B variants.

Transaldolase B (TalB) and D-fructose-6-phosphate aldolase A (FSAA) from Escherichia coli are C-C bond-forming enzymes. Using kinetic inhibition studies and mass spectrometry, it is shown that enzyme variants of FSAA and TalB that exhibit D-fructose-6-phosphate aldolase activity are inhibited covalently and irreversibly by D-tagatose 6-phosphate (D-T6P), whereas no inhibition was observed for wild-type transaldolase B from E. coli. The crystal structure of the variant TalB(F178Y) with bound sugar phosphate was solved to a resolution of 1.46 Å and revealed a novel mode of covalent inhibition. The sugar is bound covalently via its C2 atom to the ℇ-NH2 group of the active-site residue Lys132. It is neither bound in the open-chain form nor as the closed-ring form of D-T6P, but has been converted to β-D-galactofuranose 6-phosphate (D-G6P), a five-membered ring structure. The furanose ring of the covalent adduct is formed via a Heyns rearrangement and subsequent hemiacetal formation. This reaction is facilitated by Tyr178, which is proposed to act as acid-base catalyst. The crystal structure of the inhibitor complex is compared with the structure of the Schiff-base intermediate of TalB(E96Q) formed with the substrate D-fructose 6-phosphate determined to a resolution of 2.20 Å. This comparison highlights the differences in stereochemistry at the C4 atom of the ligand as an essential determinant for the formation of the inhibitor adduct in the active site of the enzyme.

CcpA Ensures Optimal Metabolic Fitness of Streptococcus pneumoniae

In Gram-positive bacteria, the transcriptional regulator CcpA is at the core of catabolite control mechanisms. In the human pathogen Streptococcus pneumoniae, links between CcpA and virulence have been established, but its role as a master regulator in different nutritional environments remains to be elucidated. Thus, we performed whole-transcriptome and metabolic analyses of S. pneumoniae D39 and its isogenic ccpA mutant during growth on glucose or galactose, rapidly and slowly metabolized carbohydrates presumably encountered by the bacterium in different host niches. CcpA affected the expression of up to 19% of the genome covering multiple cellular processes, including virulence, regulatory networks and central metabolism. Its prevalent function as a repressor was observed on glucose, but unexpectedly also on galactose. Carbohydrate-dependent CcpA regulation was also observed, as for the tagatose 6-phosphate pathway genes, which were activated by galactose and repressed by glucose. Metabolite analyses revealed that two pathways for galactose catabolism are functionally active, despite repression of the Leloir genes by CcpA. Surprisingly, galactose-induced mixed-acid fermentation apparently required CcpA, since genes involved in this type of metabolism were mostly under CcpA-repression. These findings indicate that the role of CcpA extends beyond transcriptional regulation, which seemingly is overlaid by other regulatory mechanisms. In agreement, CcpA influenced the level of many intracellular metabolites potentially involved in metabolic regulation. Our data strengthen the view that a true understanding of cell physiology demands thorough analyses at different cellular levels. Moreover, integration of transcriptional and metabolic data uncovered a link between CcpA and the association of surface molecules (e.g. capsule) to the cell wall. Hence, CcpA may play a key role in mediating the interaction of S. pneumoniae with its host. Overall, our results support the hypothesis that S. pneumoniae optimizes basic metabolic processes, likely enhancing in vivo fitness, in a CcpA-mediated manner.

Towards Enhanced Galactose Utilization by Lactococcus lactis

Accumulation of galactose in dairy products due to partial lactose fermentation by lactic acid bacteria yields poor-quality products and precludes their consumption by individuals suffering from galactosemia. This study aimed at extending our knowledge of galactose metabolism in Lactococcus lactis, with the final goal of tailoring strains for enhanced galactose consumption. We used directed genetically engineered strains to examine galactose utilization in strain NZ9000 via the chromosomal Leloir pathway (gal genes) or the plasmid-encoded tagatose 6-phosphate (Tag6P) pathway (lac genes). Galactokinase (GalK), but not galactose permease (GalP), is essential for growth on galactose. This finding led to the discovery of an alternative route, comprising a galactose phosphotransferase system (PTS) and a phosphatase, for galactose dissimilation in NZ9000. Introduction of the Tag6P pathway in a galPMK mutant restored the ability to metabolize galactose but did not sustain growth on this sugar. The latter strain was used to prove that lacFE, encoding the lactose PTS, is necessary for galactose metabolism, thus implicating this transporter in galactose uptake. Both PTS transporters have a low affinity for galactose, while GalP displays a high affinity for the sugar. Furthermore, the GalP/Leloir route supported the highest galactose consumption rate. To further increase this rate, we overexpressed galPMKT, but this led to a substantial accumulation of α-galactose 1-phosphate and α-glucose 1-phosphate, pointing to a bottleneck at the level of α-phosphoglucomutase. Overexpression of a gene encoding α-phosphoglucomutase alone or in combination with gal genes yielded strains with galactose consumption rates enhanced up to 50% relative to that of NZ9000. Approaches to further improve galactose metabolism are discussed.

Comparative functional analysis of the lac operons in Streptococcus pyogenes

Having no known environmental reservoir, Streptococcus pyogenes, a bacterium responsible for a wider variety of human diseases than any other bacterial species, must rely on its host for metabolic substrates. Although a streptococcal aldolase, LacD.1, has been adapted to virulence gene regulation, both LacD.1 and a paralogous protein, LacD.2, are predicted to function in the tagatose 6-phosphate pathway for lactose and galactose utilization. In order to gain insight into the mechanism of the LacD.1 regulatory pathway and the role of genome context in the emergence of LacD.1's novel regulatory functions, we compared the function and regulation of the Lac.1 and Lac.2 loci. The Lac.1 operon is not inducible, and regulation by LacD.1 is independent of a functional tagatose 6-phosphate pathway and enhanced by the conserved truncation of upstream Lac.1 genes. In contrast, Lac.2 expression is sensitive to environmental carbohydrates, and LacD.2, not LacD.1, contributes to growth on galactose. Thus, we conclude that the Lac.1 locus has been specialized to participate in regulation, leaving efficient utilization of carbohydrate sources to the Lac.2 locus. The adaptation of LacD for transcription regulation may be an underappreciated strategy among prokaryotes, as homologues of this multifaceted enzyme are present in a broad range of species.

Sequence, organization, transcription and regulation of lactose and galactose operons in Lactobacillus rhamnosus TCELL-1

Understanding the metabolism of lactose and galactose and their regulation in Lactobacillus rhamnosus. A gene cluster containing nine open reading frames (ORFs) involved in the metabolism of lactose and galactose in Lact. rhamnosus TCELL-1 was sequenced and characterized. The order of the ORFs was lacTEGF and galKETRM. Northern blotting experiments revealed that the gene cluster could be transcribed as one lacTEGF-galKETRM mRNA though three major transcripts (lacTEGF, galKETRM and galETRM) were detected for the gene cluster. The transcription of the lac or gal operon was independently induced in the presence of lactose or galactose. Northern blotting and primer extension experiments found the presence of four putative promoters upstream from the ORFs lacT (lacTp), galK (galKp1 and galKp2) and galE (galEp). The measurements of enzymatic activities of GalK, GalE and GalT suggested that the expression of the gal operon was subjected to a galactose activation and glucose repression mechanism. In Lact. rhamnosus TCELL-1, the galactose moiety of lactose could be metabolized by two alternative pathways (the Leloir and the tagatose 6-phosphate pathways) whereas galactose metabolism could be mediated by the Leloir pathway. This work provides important information about sugar metabolism in Lact. rhamnosus.

Lactobacillus casei 64H contains a phosphoenolpyruvate-dependent phosphotransferase system for uptake of galactose, as confirmed by analysis of ptsH and different gal mutants.

Galactose metabolism in Lactobacillus casei 64H was analyzed by genetic and biochemical methods. Mutants with defects in ptsH, galK, or the tagatose 6-phosphate pathway were isolated either by positive selection using 2-deoxyglucose or 2-deoxygalactose or by an enrichment procedure with streptozotocin. ptsH mutations abolish growth on lactose, cellobiose, N-acetylglucosamine, mannose, fructose, mannitol, glucitol, and ribitol, while growth on galactose continues at a reduced rate. Growth on galactose is also reduced, but not abolished, in galK mutants. A mutation in galK in combination with a mutation in the tagatose 6-phosphate pathway results in sensitivity to galactose and lactose, while a galK mutation in combination with a mutation in ptsH completely abolishes galactose metabolism. Transport assays, in vitro phosphorylation assays, and thin-layer chromatography of intermediates of galactose metabolism also indicate the functioning of a permease/Leloir pathway and a phosphoenolpyruvate-dependent phosphotransferase system (PTS)/tagatose 6-phosphate pathway. The galactose-PTS is induced by growth on either galactose or lactose, but the induction kinetics for the two substrates are different.

Current research on the genetics of lactic acid production in lactic acid bacteria

Abstract Lactic acid derived from lactose is a major by-product of energy production in lactic acid bacteria. The uptake of lactose by these organisms is mediated either by the lactose phosphoenolpyruvate-phosphotransferase system (lactose PEP-PTS), or by a lactose-proton symport system. The disaccharide is then converted to lactate with the concomitant production of ATP. In Lactococcus lactis the genes encoding the lactose PEP-PTS, phospho-β-galactosidase and the tagatose 6-phosphate pathway enzymes are plasmid encoded, while other genes required for lactate synthesis, including those of the Embden-Meyerhof pathway, are on the chromosome. We have compiled a current list of genes required for lactate synthesis in the lactic acid bacteria that have been cloned and characterized and discuss the present status of genetic research in this area. The analyses of the L. lactis lac operon have yielded one of the most detailed pictures of genetic regulation in this bacterium. The operon has been fully sequenced, the regulatory protein LacR which represses lac operon transcription has been purified and its properties determined, and the operon promoters and operators have been identified. Investigations of chromosomally encoded L. lactis genes have resulted in the identification and characterization of pfk, pyk, ldh, tpi and gap , which encode phosphofructokinase, pyruvate kinase, l -(+)-lactate dehydrogenase, triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase, respectively. All of these enzymes (except triosephosphate isomerase) are known from previous studies to be important in metabolite level regulation of the pathway, pfk, pyk and ldh are organized into a tricistronic operon (the las operon), while tpi and gap are in monocistronic units. The las operon is so far unique to L. lactis . A number of investigators have studied the effect of gene dosage on glycolytic flux in lactic acid bacteria and their results are reviewed. We have introduced multiple copies of pfk, pyk, Idh and the las operon into L. lactis and report the effect of the increase in gene dosage on enzyme levels and the rate of lactic acid production.

The use of bacterial luciferase genes as reporter genes in Lactococcus: regulation of the Lactococcus lactis subsp. lactis lactose genes

Lactose metabolism is an important industrial trait in dairy lactococci. In Lactococcus lactis, lactose is taken up via the phosphoenolpyruvate-dependent phosphotransferase system (PEP-PTS) and is subsequently metabolized via the glycolytic and tagatose 6-phosphate pathways. Genes for the lactose-specific PEP-PTS proteins, phospho-beta-galactosidase and tagatose 6-phosphate pathway enzymes are encoded by a single 8 kb operon, lacABCDFEGX, and there is a divergently transcribed lacR repressor gene. Transcriptional fusions of both the lac operon promoter and the lacR promoter to the luxAB genes of Vibrio fischeri were used to investigate the regulation of expression of both promoters. In vivo bioluminescence assays demonstrated that lacR negatively regulates the lac operon and also autoregulates itself. Induction of transcription occurred for both promoters during growth on lactose: sevenfold for lacR and fivefold for the lac operon. The lacR promoter was demonstrated to be a particularly strong promoter, being approximately four times more efficient than the lac operon promoter. Both promoters provide good potential for the inducible expression of foreign proteins in Lactococcus.

Streptococcus mutans serotype c tagatose 6-phosphate pathway gene cluster.

DNA cloned into Escherichia coli K-12 from a serotype c strain of Streptococcus mutans encodes three enzyme activities for galactose utilization via the tagatose 6-phosphate pathway: galactose 6-phosphate isomerase, tagatose 6-phosphate kinase, and tagatose-1,6-bisphosphate aldolase. The genes coding for the tagatose 6-phosphate pathway were located on a 3.28-kb HindIII DNA fragment. Analysis of the tagatose proteins expressed by recombinant plasmids in minicells was used to determine the sizes of the various gene products. Mutagenesis of these plasmids with transposon Tn5 was used to determine the order of the tagatose genes. Tagatose 6-phosphate isomerase appears to be composed of 14- and 19-kDa subunits. The sizes of the kinase and aldolase were found to be 34 and 36 kDa, respectively. These values correspond to those reported previously for the tagatose pathway enzymes in Staphylococcus aureus and Lactococcus lactis.

Lactose metabolism by Staphylococcus aureus: characterization of lacABCD, the structural genes of the tagatose 6-phosphate pathway.

The nucleotide and deduced amino acid sequences of the lacA and lacB genes of the Staphylococcus aureus lactose operon (lacABCDFEG) are presented. The primary translation products are polypeptides of 142 (Mr = 15,425) and 171 (Mr = 18,953) amino acids, respectively. The lacABCD loci were shown to encode enzymes of the tagatose 6-phosphate pathway through both in vitro studies and complementation analysis in Escherichia coli. A serum aldolase assay, modified to allow detection of the tagatose 6-phosphate pathway enzymes utilizing galactose 6-phosphate or fructose phosphate analogs as substrate, is described. Expression of both lacA and lacB was required for galactose 6-phosphate isomerase activity. LacC (34 kDa) demonstrated tagatose 6-phosphate kinase activity and was found to share significant homology with LacC from Lactococcus lactis and with both the minor 6-phosphofructokinase (PfkB) and 1-phosphofructokinase (FruK) from E. coli. Detection of tagatose 1,6-bisphosphate aldolase activity was dependent on expression of the 36-kDa protein specified by lacD. The LacD protein is highly homologous with LacD of L. lactis. Thus, the lacABCD genes comprise the tagatose 6-phosphate pathway and are cotranscribed with genes lacFEG, which specify proteins for transport and cleavage of lactose in S. aureus.

Molecular cloning, characterization, and nucleotide sequence of the tagatose 6-phosphate pathway gene cluster of the lactose operon of Lactococcus lactis.

The tagatose 6-phosphate pathway gene cluster (lacABCD) encoding galactose-6-phosphate isomerase, tagatose-6-phosphate kinase, and tagatose-1,6-diphosphate aldolase of Lactococcus lactis subsp. lactis MG1820 has been characterized by cloning, nucleotide sequence analysis, and enzyme assays. Transcription studies showed that the four tagatose 6-phosphate pathway genes are the first genes of the lactose-inducible lactose-phosphotransferase operon consisting of the lacABCDFEGX genes. Using a T7 expression system, it could be shown that the lacA, lacB, lacC, and lacD genes code for proteins with apparent molecular masses of 15, 19, 33, and 36 kDa, respectively. Cell-free extracts of induced and noninduced Escherichia coli cells expressing the lacABCD genes were used to determine the functions of the encoded proteins. Expression of both lacA and lacB was required to obtain galactose-6-phosphate isomerase activity. The lacC gene codes for tagatose-6-phosphate kinase, the deduced amino sequence of which is similar to that of E. coli Pfk-2 phosphofructokinase, and Staphylococcus aureus LacC protein. The tagatose-1,6-diphosphate aldolase is encoded by the lacD gene, and its deduced primary sequence, which is homologous to that of the S. aureus LacD protein, predicts an amino acid composition which is virtually identical to that of the previously purified L. lactis E8 tagatose-1,6-diphosphate aldolase.

Isolation and Characterization of Plasmid DNA in Lactobacillus acidophilus.

Lactobacillus acidophilm strain TK8912 was found to carry six pi asm ids having molecular masses of 40, 17, 10.5, 5, 2, and 1.8 megadaltons (Mdal). An acriflavin-induced, Lac- mutant had lost a 17-Mdal pi asm id (pLA102) and phospho-β-galactosidase (P-β-gal) activity. Since strain TK1-2TL (Lac+ transformant) restored P-β-gal activity, pLA102 is likely to encode a P-β-gal gene required for lactose metabolism in strain TK8912. It is also suggested that the gene for the tagatose 6-phosphate pathway, which is responsible for galactose metabolism, is encoded by the 40-Mdal plasmid pLA101.

26] Pyruvate kinase from Streptococcus lactis

Publisher Summary This chapter describes an assay method and discusses the purification procedure for pyruvate kinases from Streptococcus lactis . Pyruvate kinase activity is measured in a coupled assay system with excess lactate dehydrogenase. Oxidation of NADH is followed at 340 nm. The purification procedure involves preparation of crude extract, ammonium sulfate fractionation, chromatography on diethylaminoethyl (DEAE)-cellulose, and gel filtration on BioGel. Equilibrium sedimentation in an analytical ultracentrifuge gives a molecular weight for the purified enzyme of 235,000. The S. lactis pyruvate kinase has an obligatory requirement for both monovalent and divalent cations. K + or NH 4+ are equally effective in fulfilling the monovalent cation requirement. Streptococcus lactis pyruvate kinase is activated by a wide range of sugar mono- and diphosphates including glucose 6-phosphate, fructose 6-phosphate, and tagatose 6-phosphate. The enzyme is strongly inhibited by phosphate and to a lesser extent by sulfate, and the pH optimum under standard assay conditions in triethanolamine buffer is 7.5.

Lactose metabolism by Streptococcus mutans: evidence for induction of the tagatose 6-phosphate pathway

Growth on lactose by strains of Streptococcus mutans resulted in the induction of the lactose-phosphoenolpyruvate-phosphotransferase system, phospho-beta-galactosidase, and the enzymes of the tagatose 6-phosphate pathway.

Tagatose 6-phosphate formation from galactosamine 6-phosphate by cell-free extracts of Latcobacillus casei

Dialyzed cell-free extracts of Lactobacillus casei deaminated galactosamine 6-phosphate to tagatose 6-phosphate and ammonia.