Mannitol as a Growth Substrate for Facultative Methylotroph Methylobrevis pamukkalensis PK2.

Biochemistry of carbon assimilation in aerobic methylotrophs growing on reduced C1 compounds has been intensively studied due to the vital role of these microorganisms in nature. The biochemical pathways of carbon assimilation in methylotrophs growing on multi-carbon substrates are insufficiently explored. Here we elucidated the metabolic route of mannitol assimilation in the alphaproteobacterial facultative methylotroph Methylobrevis pamukkalensis PK2. Two key enzymes of mannitol metabolism, mannitol-2-dehydrogenase (MTD) and fructokinase (FruK), were obtained as His-tagged proteins by cloning and expression of mtd and fruK genes in Escherichia coli and characterized. Genomic analysis revealed that further transformation of fructose-6-phosphate proceeds via the Entner-Doudoroff pathway. During growth on mannitol + methanol mixture, the strain PK2 consumed both substrates simultaneously demonstrating independence of C1 and C6 metabolic pathways. Genome screening showed that genes for mannitol utilization enzymes are present in other alphaproteobacterial methylotrophs predominantly capable of living in association with plants. The capability to utilize a variety of carbohydrates (sorbitol, glucose, fructose, arabinose and xylose) suggests a broad adaptability of the strain PK2 to live in environments where availability of carbon substrate dynamically changes.

Fructose-1-kinase has pleiotropic roles in Escherichia coli.

In Escherichia coli, the master transcription regulator catabolite repressor activator (Cra) regulates >100 genes in central metabolism. Cra binding to DNA is allosterically regulated by binding to fructose-1-phosphate (F-1-P), but the only documented source of F-1-P is from the concurrent import and phosphorylation of exogenous fructose. Thus, many have proposed that fructose-1,6-bisphosphate (F-1,6-BP) is also a physiological regulatory ligand. However, the role of F-1,6-BP has been widely debated. Here, we report that the E. coli enzyme fructose-1-kinase (FruK) can carry out its "reverse" reaction under physiological substrate concentrations to generate F-1-P from F-1,6-BP. We further show that FruK directly binds Cra with nanomolar affinity and forms higher order, heterocomplexes. Growth assays with a ΔfruK strain and fruK complementation show that FruK has a broader role in metabolism than fructose catabolism. Since fruK itself is repressed by Cra, these newly-reported events add layers to the dynamic regulation of E. coli's central metabolism that occur in response to changing nutrients. These findings might have wide-spread relevance to other γ-proteobacteria, which conserve both Cra and FruK.

Ketohexokinase-A deficiency attenuates the proliferation via reducing ß-catenin in gastric cancer cells.

Overconsumption of fructose is closely related to cancer. Ketohexokinase (KHK) catalyzes the conversion from fructose to fructose-1-phosphate (F1P), which is the first and committed step of fructose metabolism. Recently, aberrant KHK activation has been identified in multiple malignancies. However, the roles of KHK in gastric cancer (GC) cells are largely unclear. Herein, we reveal that the expression of ketohexokinase-A (KHK-A), one alternatively spliced KHK isoform that possesses low affinity for fructose, was markedly increased in GC cells. Depletion of endogenous KHK-A expression using lentiviruses encoding short hairpin RNAs (shRNAs) or pharmaceutical disruption of KHK-A activity using KHK-IN-1 hydrochloride in GC NCI-N87 and HGC-27 cells inhibited the proliferation in vitro and in vivo. Additionally, the mitochondrial respiration in the GC cells with KHK-A deficiency compared with the control cells was significantly impaired. One commercially-available antibody array was used to explore the effects of KHK-A knockdown on signaling pathways, showing that ß-catenin was remarkably reduced in the KHK-A deficient GC cells compared with the control ones. Pharmaceutical reduction in ß-catenin levels slowed down the proliferation of GC cells. These data uncover that KHK-A promotes the proliferation in GC cells, indicating that this enzyme might be a promising therapeutical target for GC treatment.

The polyol pathway and nuclear ketohexokinase A signaling drive hyperglycemia-induced metastasis of gastric cancer.

Diabetes might be associated with increased cancer risk, with several studies reporting hyperglycemia as a primary oncogenic stimulant. Since glucose metabolism is linked to numerous metabolic pathways, it is difficult to specify the mechanisms underlying hyperglycemia-induced cancer progression. Here, we focused on the polyol pathway, which is dramatically activated under hyperglycemia and causes diabetic complications. We investigated whether polyol pathway-derived fructose facilitates hyperglycemia-induced gastric cancer metastasis. We performed bioinformatics analysis of gastric cancer datasets and immunohistochemical analyses of gastric cancer specimens, followed by transcriptomic and proteomic analyses to evaluate phenotypic changes in gastric cancer cells. Consequently, we found a clinical association between the polyol pathway and gastric cancer progression. In gastric cancer cell lines, hyperglycemia enhanced cell migration and invasion, cytoskeletal rearrangement, and epithelial-mesenchymal transition (EMT). The hyperglycemia-induced acquisition of metastatic potential was mediated by increased fructose derived from the polyol pathway, which stimulated the nuclear ketohexokinase-A (KHK-A) signaling pathway, thereby inducing EMT by repressing the CDH1 gene. In two different xenograft models of cancer metastasis, gastric cancers overexpressing AKR1B1 were found to be highly metastatic in diabetic mice, but these effects of AKR1B1 were attenuated by KHK-A knockdown. In conclusion, hyperglycemia induces fructose formation through the polyol pathway, which in turn stimulates the KHK-A signaling pathway, driving gastric cancer metastasis by inducing EMT. Thus, the polyol and KHK-A signaling pathways could be potential therapeutic targets to decrease the metastatic risk in gastric cancer patients with diabetes.