Lactate dehydrogenase A regulates tumor-macrophage symbiosis to promote glioblastoma progression.

Abundant macrophage infiltration and altered tumor metabolism are two key hallmarks of glioblastoma. By screening a cluster of metabolic small-molecule compounds, we show that inhibiting glioblastoma cell glycolysis impairs macrophage migration and lactate dehydrogenase inhibitor stiripentol emerges as the top hit. Combined profiling and functional studies demonstrate that lactate dehydrogenase A (LDHA)-directed extracellular signal-regulated kinase (ERK) pathway activates yes-associated protein 1 (YAP1)/ signal transducer and activator of transcription 3 (STAT3) transcriptional co-activators in glioblastoma cells to upregulate C-C motif chemokine ligand 2 (CCL2) and CCL7, which recruit macrophages into the tumor microenvironment. Reciprocally, infiltrating macrophages produce LDHA-containing extracellular vesicles to promote glioblastoma cell glycolysis, proliferation, and survival. Genetic and pharmacological inhibition of LDHA-mediated tumor-macrophage symbiosis markedly suppresses tumor progression and macrophage infiltration in glioblastoma mouse models. Analysis of tumor and plasma samples of glioblastoma patients confirms that LDHA and its downstream signals are potential biomarkers correlating positively with macrophage density. Thus, LDHA-mediated tumor-macrophage symbiosis provides therapeutic targets for glioblastoma.

LRPPRC promotes glycolysis by stabilising LDHA mRNA and its knockdown plus glutamine inhibitor induces synthetic lethality via m6 A modification in triple-negative breast cancer.

BACKGROUND: Targeted therapy for triple-negative breast cancer (TNBC) remains a challenge. N6-methyladenosine (m6 A) is the most abundant internal mRNA modification in eukaryotes, and it regulates the homeostasis and function of modified RNA transcripts in cancer. However, the role of leucine-rich pentatricopeptide repeat containing protein (LRPPRC) as an m6 A reader in TNBC remains poorly understood. METHODS: Western blotting, reverse transcription-polymerase chain reaction (RT-qPCR) and immunohistochemistry were used to investigate LRPPRC expression levels. Dot blotting and colorimetric enzyme linked immunosorbent assay (ELISA) were employed to detect m6 A levels. In vitro functional assays and in vivo xenograft mouse model were utilised to examine the role of LRPPRC in TNBC progression. Liquid chromatography-mass spectrometry/mass spectrometry and Seahorse assays were conducted to verify the effect of LRPPRC on glycolysis. MeRIP-sequencing, RNA-sequencing, MeRIP assays, RNA immunoprecipitation assays, RNA pull-down assays and RNA stability assays were used to identify the target genes of LRPPRC. Patient-derived xenografts and organoids were employed to substantiate the synthetic lethality induced by LRPPRC knockdown plus glutaminase inhibition. RESULTS: The expressions of LRPPRC and m6 A RNA were elevated in TNBC, and the m6 A modification site could be recognised by LRPPRC. LRPPRC promoted the proliferation, metastasis and glycolysis of TNBC cells both in vivo and in vitro. We identified lactate dehydrogenase A (LDHA) as a novel direct target of LRPPRC, which recognised the m6 A site of LDHA mRNA and enhanced the stability of LDHA mRNA to promote glycolysis. Furthermore, while LRPPRC knockdown reduced glycolysis, glutaminolysis was enhanced. Moreover, the effect of LRPPRC on WD40 repeat domain-containing protein 76 (WDR76) mRNA stability was impaired in an m6 A-dependent manner. Then, LRPPRC knockdown plus a glutaminase inhibition led to synthetic lethality. CONCLUSIONS: Our study demonstrated that LRPPRC promoted TNBC progression by regulating metabolic reprogramming via m6 A modification. These characteristics shed light on the novel combination targeted therapy strategies to combat TNBC.

The catalytic action of human d-lactate dehydrogenase is severely inhibited by oxalate and is impaired by mutations triggering d-lactate acidosis.

d-lactate dehydrogenases are known to be expressed by prokaryotes and by eukaryotic invertebrates, and over the years the functional and structural features of some bacterial representatives of this enzyme ensemble have been investigated quite in detail. Remarkably, a human gene coding for a putative d-lactate dehydrogenase (DLDH) was identified and characterized, disclosing the occurrence of alternative splicing of its primary transcript. This translates into the expression of two human DLDH (hDLDH) isoforms, the molecular mass of which is expected to differ by 2.7 kDa. However, no information on these two hDLDH isoforms is available at the protein level. Here we report on the catalytic action of these enzymes, along with a first analysis of their structural features. In particular, we show that hDLDH is strictly stereospecific, with the larger isoform (hDLDH-1) featuring higher activity at the expense of d-lactate when compared to its smaller counterpart (hDLDH-2). Furthermore, we found that hDLDH is strongly inhibited by oxalate, as indicated by a Ki equal to 1.2 µM for this dicarboxylic acid. Structurally speaking, hDLDH-1 and hDLDH-2 were determined, by means of gel filtration and dynamic light scattering experiments, to be a hexamer and a tetramer, respectively. Moreover, in agreement with previous studies performed with human mitochondria, we identified FAD as the cofactor of hDLDH, and we report here a model of FAD binding by the human d-lactate dehydrogenase. Interestingly, the mutations W323C and T412 M negatively affect the activity of hDLDH, most likely by impairing the enzyme electron-acceptor site.

Palmitoylation alters LDHA activity and pancreatic cancer response to chemotherapy.

Lactate dehydrogenase A (LDHA) serves as a key regulator of the Warburg Effect by catalyzing the conversion of pyruvate to lactate in the final step of glycolysis. Both the expression level and enzyme activity of LDHA are upregulated in cancers, however, the underlying mechanism remains incompletely understood. Here, we show that LDHA is post-translationally palmitoylated by ZDHHC9 at cysteine 163, which promotes its enzyme activity, lactate production, and reduces reactive oxygen species (ROS) generation. Replacement of endogenous LDHA with a palmitoylation-deficient mutant leads to reduced pancreatic cancer cell proliferation, increased T-cell infiltration, and limited tumor growth; it also affects pancreatic cancer cell response to chemotherapy. Moreover, LDHA palmitoylation is upregulated in gemcitabine resistant pancreatic cancer cells. Clinically, ZDHHC9 is upregulated in pancreatic cancer and correlated with poor prognoses for patients. Overall, our findings identify ZDHHC9-mediated palmitoylation as a positive regulator of LDHA, with potentially significant implications for cancer etiology and targeted therapy for pancreatic cancer.

Pyramiding D-lactate dehydrogenase with the glyoxalase pathway enhances abiotic stress tolerance in plants.

Methylglyoxal is a common cytotoxic metabolite produced in plants during multiple biotic and abiotic stress. To mitigate the toxicity of MG, plants utilize the glyoxalase pathway comprising glyoxalase I (GLYI), glyoxalase II (GLYII), or glyoxalase III (GLYIII). GLYI and GLYII are the key enzymes of glyoxalase pathways that play an important role in abiotic stress tolerance. Earlier research showed that MG level is lower when both GLYI and GLYII are overexpressed together, compared to GLYI or GLYII single gene overexpressed transgenic plants. D-lactate dehydrogenase (D-LDH) is an integral part of MG detoxification which metabolizes the end product (D-lactate) of the glyoxalase pathway. In this study, two Arabidopsis transgenic lines were constructed using gene pyramiding technique: GLYI and GLYII overexpressed (G-I + II), and GLYI, GLYII, and D-LDH overexpressed (G-I + II + D) plants. G-I + II + D exhibits lower MG and D-lactate levels and enhanced abiotic stress tolerance than the G-I + II and wild-type plants. Further study explores the stress tolerance mechanism of G-I + II + D plants through the interplay of different regulators and plant hormones. This, in turn, modulates the expression of ABA-dependent stress-responsive genes like RAB18, RD22, and RD29B to generate adaptive responses during stress. Therefore, there might be a potential correlation between ABA and MG detoxification pathways. Furthermore, higher STY46, GPX3, and CAMTA1 transcripts were observed in G-I + II + D plants during abiotic stress. Thus, our findings suggest that G-I + II + D has significantly improved MG detoxification, reduced oxidative stress-induced damage, and provided a better protective mechanism against abiotic stresses than G-I + II or wild-type plants.

Causal relationship between lactate dehydrogenase and risk of developing ischemic stroke: A Mendelian randomized study.

BACKGROUND AND OBJECTIVE: Ischemic stroke (IS) is one of the major global health problems. It is not clear whether there is a causal relationship between lactate dehydrogenase (LDH) and the risk of IS attacks. The purpose of this study was to investigate whether LDH has a causal relationship with the development of IS. METHODS: The genome-wide association data of LDH and IS were obtained through a Mendelian randomization-based platform. Single nucleotide polymorphisms (SNP) that were significantly associated with LDH were identified and used as instrumental variables, and a two-sample Mendelian randomization study was used to examine the causal relationship between LDH and IS. The statistical methods included Inverse-variance weighted approach, MR-Egger regression, and weighted median estimator. RESULTS: We selected 15 SNPs of genome-wide significance from Genome-wide association study database with LDH as instrumental variables. A consistent causal association between LDH and IS was observed by different assessment methods. The results of the inverse-variance weighted method suggested an inverse association between LDH and higher genetic predictability of IS risk (OR, 0.997; 95%CI 0.995-0.999). The weighted median estimate showed consistent results with the MR-Egger method (weighted median estimate: OR, 0.995; 95%CI 0.992-0.999; MR-Egger method: OR, 0.996; 95%CI 0.992-0.999). The inverse-variance weighted method indicates a causal association between LDH and IS (ß = -0.002563, SE = 0.00128, p = .0453). MR-Egger analysis (ß = -0.004498, SE = 0.001877, p = .03) and the weighted median method suggested that LDH and IS also existed causal relationship (ß = -0.004861, SE = 0.001801, p = .00695). CONCLUSIONS: Our Mendelian randomization results suggest that LDH is inversely associated with the risk of developing IS, and are contrary to the results of previous observational studies.

Enhanced catalytic activity and stability of lactate dehydrogenase for cascade catalysis of D-PLA by rational design.

Serving as a vital medical intermediate and an environmentally-friendly preservative, D-PLA exhibits substantial potential across various industries. In this report, the urgent need for efficient production motivated us to achieve the rational design of lactate dehydrogenase and enhance catalytic efficiency. Surprisingly, the enzymatic properties revealed that a mutant enzyme, LrLDHT247I/D249A/F306W/A214Y (LrLDH-M1), had a viable catalytic advantage. It demonstrated a 3.3-fold increase in specific enzyme activity and approximately a 2.08-fold improvement of Kcat. Correspondingly, molecular docking analysis provided a supporting explanation for the lower Km and higher Kcat/Km of the mutant enzyme. Thermostability analysis exhibited increased half-lives and the deactivation rate constants decreased at different temperatures (1.47-2.26-fold). In addition, the mutant showed excellent resistance abilities in harsh environments, particularly under acidic conditions. Then, a two-bacterium (E. coli/pET28a-lrldh-M1 and E. coli/pET28a-ladd) coupled catalytic system was developed and realized a significant conversion rate (77.7%) of D-phenyllactic acid, using 10 g/L L-phenylalanine as the substrate in a two-step cascade reaction.

Forward Genetic Screens Identify Mechanisms of Resistance to Small-Molecule Lactate Dehydrogenase Inhibitors.

Altered metabolism is a hallmark of cancer; however, it has been difficult to specifically target metabolism in cancer for therapeutic benefit. Cancers with genetically defined defects in metabolic enzymes constitute a subset of cancers where targeting metabolism is potentially accessible. Hürthle cell carcinoma of the thyroid (HTC) tumors frequently harbor deleterious mitochondrial DNA (mtDNA) mutations in subunits of complex I of the mitochondrial electron transport chain (ETC). Previous work has shown that HTC models with deleterious mtDNA mutations exhibit mitochondrial ETC defects that expose lactate dehydrogenase (LDH) as a therapeutic vulnerability. Here, we performed forward genetic screens to identify mechanisms of resistance to small-molecule LDH inhibitors. We identified two distinct mechanisms of resistance: upregulation of an LDH isoform and a compound-specific resistance mutation. Using these tools, we demonstrate that the anticancer activity of LDH inhibitors in cell line and xenograft models of complex I mutant HTC is through on-target LDH inhibition.

Limited threat of Plasmodium falciparum pfhrp2 and pfhrp3 gene deletion to the utility of HRP2-based malaria RDTs in Northern Uganda.

BACKGROUND: Rapid diagnostic tests (RDTs) that detect Plasmodium falciparum histidine-rich protein-2 (PfHRP2) are exclusively deployed in Uganda, but deletion of the pfhrp2/3 target gene threatens their usefulness as malaria diagnosis and surveillance tools. METHODS: A cross-sectional survey was conducted at 40 sites across four regions of Uganda in Acholi, Lango, W. Nile and Karamoja from March 2021 to June 2023. Symptomatic malaria suspected patients were recruited and screened with both HRP2 and pan lactate dehydrogenase (pLDH) detecting RDTs. Dried blood spots (DBS) were collected from all patients and a random subset were used for genomic analysis to confirm parasite species and pfhrp2 and pfhrp3 gene status. Plasmodium species was determined using a conventional multiplex PCR while pfhrp2 and pfhrp3 gene deletions were determined using a real-time multiplex qPCR. Expression of the HRP2 protein antigen in a subset of samples was further assessed using a ELISA. RESULTS: Out of 2435 symptomatic patients tested for malaria, 1504 (61.8%) were positive on pLDH RDT. Overall, qPCR confirmed single pfhrp2 gene deletion in 1 out of 416 (0.2%) randomly selected samples that were confirmed of P. falciparum mono-infections. CONCLUSION: These findings show limited threat of pfhrp2/3 gene deletions in the survey areas suggesting that HRP2 RDTs are still useful diagnostic tools for surveillance and diagnosis of P. falciparum malaria infections in symptomatic patients in this setting. Periodic genomic surveillance is warranted to monitor the frequency and trend of gene deletions and its effect on RDTs.

Characterization of genetic and molecular tools for studying the endogenous expression of Lactate dehydrogenase in Drosophila melanogaster.

Drosophila melanogaster larval development relies on a specialized metabolic state that utilizes carbohydrates and other dietary nutrients to promote rapid growth. One unique feature of the larval metabolic program is that Lactate Dehydrogenase (Ldh) activity is highly elevated during this growth phase when compared to other stages of the fly life cycle, indicating that Ldh serves a key role in promoting juvenile development. Previous studies of larval Ldh activity have largely focused on the function of this enzyme at the whole animal level, however, Ldh expression varies significantly among larval tissues, raising the question of how this enzyme promotes tissue-specific growth programs. Here we characterize two transgene reporters and an antibody that can be used to study Ldh expression in vivo. We find that all three tools produce similar Ldh expression patterns. Moreover, these reagents demonstrate that the larval Ldh expression pattern is complex, suggesting the purpose of this enzyme varies across cell types. Overall, our studies validate a series of genetic and molecular reagents that can be used to study glycolytic metabolism in the fly.

OCT1 regulates the migration of colorectal cancer cells by acting on LDHA.

Colorectal cancer is one of the most common cancers with high morbidity and mortality. Effective treatments to improve the prognosis are still lacking. The results of online analysis tools showed that OCT1 and LDHA were highly expressed in colorectal cancer, and the high expression of OCT1 was associated with poor prognosis. Immunofluorescence demonstrated that OCT1 and LDHA co-localized in colorectal cancer cells. In colorectal cancer cells, OCT1 and LDHA were upregulated by OCT1 overexpression, but downregulated by OCT1 knockdown. OCT1 overexpression promoted cell migration. OCT1 or LDHA knockdown inhibited the migration, and the downregulation of LDHA restored the promoting effect of OCT1 overexpression. OCT1 upregulation increased the levels of HK2, GLUT1 and LDHA proteins in colorectal cancer cells. Consequently, OCT1 promoted the migration of colorectal cancer cells by upregulating LDHA.

Comprehensive evaluation of recombinant lactate dehydrogenase production from inclusion bodies.

A broad application spectrum ranging from clinical diagnostics to biosensors in a variety of sectors, makes the enzyme Lactate dehydrogenase (LDH) highly interesting for recombinant protein production. Expression of recombinant LDH is currently mainly carried out in uncontrolled shake-flask cultivations leading to protein that is mostly produced in its soluble form, however in rather low yields. Inclusion body (IB) processes have gathered a lot of attention due to several benefits like increased space-time yields and high purity of the target product. Thus, to investigate the suitability of this processing strategy for ldhL1 production, a fed-batch fermentation steering the production of IBs rather than soluble product formation was developed. It was shown that the space-time-yield of the fermentation could be increased almost 3-fold by increasing qs to 0.25 g g-1 h-1 which corresponds to 21% of qs,max, and keeping the temperature at 37°C after induction. Solubilization and refolding unit operations were developed to regain full bioactivity of the ldhL1. The systematic approach in screening for solubilization and refolding conditions revealed buffer compositions and processing strategies that ultimately resulted in 50% product recovery in the refolding step, revealing major optimization potential in the downstream processing chain. The recovered ldhL1 showed an optimal activity at pH 5.5 and 30∘C with a high catalytic activity and KM values of 0.46 mM and 0.18 mM for pyruvate and NADH, respectively. These features, show that the here produced LDH is a valuable source for various commercial applications, especially considering low pH-environments.

Rapid diagnostic tests for malaria diagnosis in Cameroon: impact of histidine rich protein 2/3 deletions and lactate dehydrogenase gene polymorphism.

Malaria rapid diagnostic tests (mRDT) play a vital role in malaria control in endemic areas. In this study, histidine-rich protein (hrp) and lactate dehydrogenase (ldh) genes were genotyped in Plasmodium falciparum (Pf) and Plasmodium ovale (Po) spp. isolates. Deletions in P. falciparum hrp2/3 (pfhrp2/3) proteins and single nucleotide polymorphisms (SNPs) were analyzed. Twenty-four samples were analyzed for pfhrp2/3 gene deletions and 25 for SNPs in ldh gene (18 Pf and 7 Po spp.). Deletions in pfhrp2/3 genes were observed in 1.9% malaria positive isolates. The pfldh gene sequences showed one SNP at codon 272 (D272N) in 22.2% of samples while in Po spp., sequences were 100% similar to P. ovale curtisi but when compared to P. ovale wallikeri reference sequence, SNPs at positions 143 (P143S), 168 (K168N), 204 (V204I) were found. Findings suggest low prevalence in pfhrp2/3 genes and highlight the circulation of P. ovale curtisi in the studies areas.

HIF-1-mediated regulation of LDH gene unravels key insights into MCDV-1 pathogenesis in mud crabs Scylla paramamosain.

Hypoxia-inducible factors -1 (HIF-1) is a crucial transcription factor that regulates the expression of glycolytic genes. Our previous study proved that the Mud crab dicistrovirus-1 (MCDV-1) can induce aerobic glycolysis that favors viral replication in mud crab Scylla paramamosain. However, the role of HIF-1 on key glycolytic genes during the MCDV-1 infection has not been examined. In this study, the intricate interplay between HIF-1 and the key glycolysis enzyme, lactate dehydrogenase (LDH), was investigated after MCDV-1 infection. The expression of LDH was significant increased after MCDV-1 infection. Additionally, the expression of HIF-1α was upregulated following MCDV-1 infection, potentially attributed to the downregulation of prolyl hydroxylase domains 2 expression. Subsequent examination of the SpLDH promoter identified the presence of hypoxia response elements (HREs), serving as binding sites for HIF-1α. Intriguingly, experimental evidence demonstrated that SpHIF-1α actively promotes SpLDH transcription through these HREs. To further elucidate the functional significance of SpHIF-1α, targeted silencing was employed, resulting in a substantial reduction in SpLDH expression, activity, and lactate concentrations in MCDV-1-infected mud crabs. Notably, SpHIF-1α-silenced mud crabs exhibited higher survival rates and lower viral loads in hepatopancreas tissues following MCDV-1 infection. These results highlight the critical role of SpHIF-1α in MCDV-1 pathogenesis by regulating LDH gene dynamics, providing valuable insights into the molecular mechanisms underlying the virus-host interaction.

Deciphering Evolutionary Trajectories of Lactate Dehydrogenases Provides New Insights into Allostery.

Lactate dehydrogenase (LDH, EC.1.1.127) is an important enzyme engaged in the anaerobic metabolism of cells, catalyzing the conversion of pyruvate to lactate and NADH to NAD+. LDH is a relevant enzyme to investigate structure-function relationships. The present work provides the missing link in our understanding of the evolution of LDHs. This allows to explain (i) the various evolutionary origins of LDHs in eukaryotic cells and their further diversification and (ii) subtle phenotypic modifications with respect to their regulation capacity. We identified a group of cyanobacterial LDHs displaying eukaryotic-like LDH sequence features. The biochemical and structural characterization of Cyanobacterium aponinum LDH, taken as representative, unexpectedly revealed that it displays homotropic and heterotropic activation, typical of an allosteric enzyme, whereas it harbors a long N-terminal extension, a structural feature considered responsible for the lack of allosteric capacity in eukaryotic LDHs. Its crystallographic structure was solved in 2 different configurations typical of the R-active and T-inactive states encountered in allosteric LDHs. Structural comparisons coupled with our evolutionary analyses helped to identify 2 amino acid positions that could have had a major role in the attenuation and extinction of the allosteric activation in eukaryotic LDHs rather than the presence of the N-terminal extension. We tested this hypothesis by site-directed mutagenesis. The resulting C. aponinum LDH mutants displayed reduced allosteric capacity mimicking those encountered in plants and human LDHs. This study provides a new evolutionary scenario of LDHs that unifies descriptions of regulatory properties with structural and mutational patterns of these important enzymes.

Lactate as a major epigenetic carbon source for histone acetylation via nuclear LDH metabolism.

Histone acetylation involves the transfer of two-carbon units to the nucleus that are embedded in low-concentration metabolites. We found that lactate, a high-concentration metabolic byproduct, can be a major carbon source for histone acetylation through oxidation-dependent metabolism. Both in cells and in purified nuclei, 13C3-lactate carbons are incorporated into histone H4 (maximum incorporation: ~60%). In the purified nucleus, this process depends on nucleus-localized lactate dehydrogenase (LDHA), knockout (KO) of which abrogates incorporation. Heterologous expression of nucleus-localized LDHA reverses the KO effect. Lactate itself increases histone acetylation, whereas inhibition of LDHA reduces acetylation. In vitro and in vivo settings exhibit different lactate incorporation patterns, suggesting an influence on the microenvironment. Higher nuclear LDHA localization is observed in pancreatic cancer than in normal tissues, showing disease relevance. Overall, lactate and nuclear LDHA can be major structural and regulatory players in the metabolism-epigenetics axis controlled by the cell's own status or the environmental status.

[Effects of manipulating lactate dehydrogenase gene on metabolism of HEK-293 and production of human adenovirus].

Reducing lactate accumulation has always been a goal of the mammalian cell biotechnology industry. When animal cells are cultured in vitro, the accumulation of lactate is mainly the combined result of two metabolic pathways. On one hand, glucose generates lactate under the function of lactate dehydrogenase A (LDHA); on the other hand, lactate can be oxidized to pyruvate by LDHB or LDHC and re-enter the TCA cycle. This study comprehensively evaluated the effects of LDH manipulation on the growth, metabolism and human adenovirus (HAdV) production of human embryonic kidney 293 (HEK-293) cells, providing a theoretical basis for engineering the lactate metabolism in mammalian cells. By knocking out ldha gene and overexpression of ldhb and ldhc genes, the metabolic efficiency of HEK-293 cells was effectively improved, and HAdV production was significantly increased. Compared with the control cell, LDH manipulation promoted cell growth, reduced the accumulation of lactate and ammonia, significantly enhanced the efficiency of substrate and energy metabolism of cells, and significantly increased the HAdV production capacity of HEK-293 cells. Among these LDH manipulation measures, ldhc gene overexpression performed the best, with the maximum cell density increased by about 38.7%. The yield of lactate to glucose and ammonia to glutamine decreased by 33.8% and 63.3%, respectively; and HAdV titer increased by at least 16 times. In addition, the ATP production rate, ATP/O2 ratio, ATP/ADP ratio and NADH content of the modified cell lines were increased to varying degrees, and the energy metabolic efficiency was significantly improved.

Metagenomic insights into the microbiota involved in lactate and butyrate production and manipulating their synthesis in alfalfa silage.

AIMS: Lactate and butyrate are important indicators of silage quality. However, the microorganisms and mechanisms responsible for lactate and butyrate production in silage are not well documented. METHODS AND RESULTS: whole-metagenomic sequencing was used to analyse metabolic pathways, microbiota composition, functional genes, and their contributions to lactate and butyrate production in alfalfa silage with (SA) and without (CK) sucrose addition. Carbon metabolism was the most abundant metabolic pathway. We identified 11 and 2 functional genes associated with lactate and butyrate metabolism, respectively. Among them, D-lactate dehydrogenase (ldhA) and L-lactate dehydrogenase (ldhB) were most important for the transition between D/L-lactate and pyruvate and were primarily related to Lactobacillus in the SA group. The genes encoding L-lactate dehydrogenase (lldD), which decomposes lactate, were the most abundant and primarily associated with Enterobacter cloacae. Butyrate-related genes, mainly encoding butyryl-CoA: acetate CoA-transferase (but), were predominantly associated with Klebsiella oxytoca and Escherichia coli in the CK group. CONCLUSIONS: Enterobacteriaceae and Lactobacillaceae were mainly responsible for butyrate and lactate formation, respectively.

Characterization of the Pro101Gln mutation that enhances the catalytic performance of T. indicus NADH-dependent d-lactate dehydrogenase.

NADH-dependent d-lactate dehydrogenases (d-LDH) are important for the industrial production of d-lactic acid. Here, we identify and characterize an improved d-lactate dehydrogenase mutant (d-LDH1) that contains the Pro101Gln mutation. The specific enzyme activities of d-LDH1 toward pyruvate and NADH are 21.8- and 11.0-fold greater compared to the wild-type enzyme. We determined the crystal structure of Apo-d-LDH1 at 2.65 Å resolution. Based on our structural analysis and docking studies, we explain the differences in activity with an altered binding conformation of NADH in d-LDH1. The role of the conserved residue Pro101 in d-LDH was further probed in site-directed mutagenesis experiments. We introduced d-LDH1 into Bacillus licheniformis yielding a d-lactic acid production of 145.9 g L-1 within 60 h at 50°C, which was three times higher than that of the wild-type enzyme. The discovery of d-LDH1 will pave the way for the efficient production of d-lactic acid by thermophilic bacteria.

The Ratio of Key Metabolic Transcripts Is a Predictive Biomarker of Breast Cancer Metastasis to the Lung.

Understanding the rewired metabolism underlying organ-specific metastasis in breast cancer could help identify strategies to improve the treatment and prevention of metastatic disease. Here, we used a systems biology approach to compare metabolic fluxes used by parental breast cancer cells and their brain- and lung-homing derivatives. Divergent lineages had distinct, heritable metabolic fluxes. Lung-homing cells maintained high glycolytic flux despite low levels of glycolytic intermediates, constitutively activating a pathway sink into lactate. This strong Warburg effect was associated with a high ratio of lactate dehydrogenase (LDH) to pyruvate dehydrogenase (PDH) expression, which correlated with lung metastasis in patients with breast cancer. Although feature classification models trained on clinical characteristics alone were unable to predict tropism, the LDH/PDH ratio was a significant predictor of metastasis to the lung but not to other organs, independent of other transcriptomic signatures. High lactate efflux was also a trait in lung-homing metastatic pancreatic cancer cells, suggesting that lactate production may be a convergent phenotype in lung metastasis. Together, these analyses highlight the essential role that metabolism plays in organ-specific cancer metastasis and identify a putative biomarker for predicting lung metastasis in patients with breast cancer. SIGNIFICANCE: Lung-homing metastatic breast cancer cells express an elevated ratio of lactate dehydrogenase to pyruvate dehydrogenase, indicating that ratios of specific metabolic gene transcripts have potential as metabolic biomarkers for predicting organ-specific metastasis.