Hydroxysteroid 17-ß dehydrogenase 14 (HSD17B14) is an L-fucose dehydrogenase, the initial enzyme of the L-fucose degradation pathway.

L-Fucose (6-deoxy-L-galactose), a monosaccharide abundant in glycolipids and glycoproteins produced by mammalian cells, has been extensively studied for its role in intracellular biosynthesis and recycling of GDP-L-fucose for fucosylation. However, in certain mammalian species, L-fucose is efficiently broken down to pyruvate and lactate in a poorly understood metabolic pathway. In the 1970s, L-fucose dehydrogenase, an enzyme responsible for the initial step of this pathway, was partially purified from pig and rabbit livers and characterized biochemically. However, its molecular identity remained elusive until recently. This study reports the purification, identification, and biochemical characterization of the mammalian L-fucose dehydrogenase. The enzyme was purified from rabbit liver approximately 340-fold. Mass spectrometry analysis of the purified protein preparation identified mammalian hydroxysteroid 17-ß dehydrogenase 14 (HSD17B14) as the sole candidate enzyme. Rabbit and human HSD17B14 were expressed in HEK293T and Escherichia coli, respectively, purified and demonstrated to catalyze the oxidation of L-fucose to L-fucono-1,5-lactone, as confirmed by mass spectrometry and NMR analysis. Substrate specificity studies revealed that L-fucose is the preferred substrate for both enzymes. The human enzyme exhibited a catalytic efficiency for L-fucose that was 359-fold higher than its efficiency for estradiol. Additionally, recombinant rat HSD17B14 exhibited negligible activity towards L-fucose, consistent with the absence of L-fucose metabolism in this species. The identification of the gene encoding mammalian L-fucose dehydrogenase provides novel insights into the substrate specificity of enzymes belonging to the 17-ß-hydroxysteroid dehydrogenase family. This discovery also paves the way for unraveling the physiological functions of the L-fucose degradation pathway, which remains enigmatic.

The Role of Trp79 in ß-Actin on Histidine Methyltransferase SETD3 Catalysis.

Nτ -methylation of His73 in actin by histidine methyltransferase SETD3 plays an important role in stabilising actin filaments in eukaryotes. Mutations in actin and overexpression of SETD3 have been related to human diseases, including cancer. Here, we investigated the importance of Trp79 in ß-actin on productive human SETD3 catalysis. Substitution of Trp79 in ß-actin peptides by its chemically diverse analogues reveals that the hydrophobic Trp79 binding pocket modulates the catalytic activity of SETD3, and that retaining a bulky and hydrophobic amino acid at position 79 is important for efficient His73 methylation by SETD3. Molecular dynamics simulations show that the Trp79 binding pocket of SETD3 is ideally shaped to accommodate large and hydrophobic Trp79, contributing to the favourable release of water molecules upon binding. Our results demonstrate that the distant Trp79 binding site plays an important role in efficient SETD3 catalysis, contributing to the identification of new SETD3 substrates and the development of chemical probes targeting the biomedically important SETD3.

Histidine methyltransferase SETD3 methylates structurally diverse histidine mimics in actin.

Actin histidine Nτ -methylation by histidine methyltransferase SETD3 plays an important role in human biology and diseases. Here, we report integrated synthetic, biocatalytic, biostructural, and computational analyses on human SETD3-catalyzed methylation of actin peptides possessing histidine and its structurally and chemically diverse mimics. Our enzyme assays supported by biostructural analyses demonstrate that SETD3 has a broader substrate scope beyond histidine, including N-nucleophiles on the aromatic and aliphatic side chains. Quantum mechanical/molecular mechanical molecular dynamics and free-energy simulations provide insight into binding geometries and the free energy barrier for the enzymatic methyl transfer to histidine mimics, further supporting experimental data that histidine is the superior SETD3 substrate over its analogs. This work demonstrates that human SETD3 has a potential to catalyze efficient methylation of several histidine mimics, overall providing mechanistic, biocatalytic, and functional insight into actin histidine methylation by SETD3.

Importance of Ile71 in ß-actin on histidine methyltransferase SETD3 catalysis.

SETD3-catalysed N3-methylation of His73 in ß-actin plays a key role in stabilisation of actin filaments in the metazoan cells. Overexpression and/or dysregulation of SETD3 is associated with several human pathologies, including cancer. Here, we examined the role of the Ile71 residue in ß-actin on human SETD3 catalysis. Substitution of Ile71 in ß-actin peptides by its natural and unnatural mimics reveals that the 'secondary' Ile71 binding pocket modulates the substrate efficiency of ß-actin. Our enzymatic work demonstrates that human SETD3 can accommodate structurally diverse hydrophobic side chains in its Ile71 binding pocket, providing clear limits of the size and shape of Ile analogues. Water thermodynamics calculations reveal that the Ile71 pocket is occupied by high-energy water molecules, that are released upon the Ile71 binding, contributing favourably to the SETD3-ßA complex formation. The work highlights that the hydrophobic Ile71 binding site plays an essential role in SETD3 catalysis, contributing to an ongoing effort in the design and development of chemical probes targeting SETD3.

Recharacterization of the mammalian cytosolic type 2 (R)-ß-hydroxybutyrate dehydrogenase as 4-oxo-l-proline reductase (EC 1.1.1.104).

Early studies revealed that chicken embryos incubated with a rare analog of l-proline, 4-oxo-l-proline, showed increased levels of the metabolite 4-hydroxy-l-proline. In 1962, 4-oxo-l-proline reductase, an enzyme responsible for the reduction of 4-oxo-l-proline, was partially purified from rabbit kidneys and characterized biochemically. However, only recently was the molecular identity of this enzyme solved. Here, we report the purification from rat kidneys, identification, and biochemical characterization of 4-oxo-l-proline reductase. Following mass spectrometry analysis of the purified protein preparation, the previously annotated mammalian cytosolic type 2 (R)-ß-hydroxybutyrate dehydrogenase (BDH2) emerged as the only candidate for the reductase. We subsequently expressed rat and human BDH2 in Escherichia coli, then purified it, and showed that it catalyzed the reversible reduction of 4-oxo-l-proline to cis-4-hydroxy-l-proline via chromatographic and tandem mass spectrometry analysis. Specificity studies with an array of compounds carried out on both enzymes showed that 4-oxo-l-proline was the best substrate, and the human enzyme acted with 12,500-fold higher catalytic efficiency on 4-oxo-l-proline than on (R)-ß-hydroxybutyrate. In addition, human embryonic kidney 293T (HEK293T) cells efficiently metabolized 4-oxo-l-proline to cis-4-hydroxy-l-proline, whereas HEK293T BDH2 KO cells were incapable of producing cis-4-hydroxy-l-proline. Both WT and KO HEK293T cells also produced trans-4-hydroxy-l-proline in the presence of 4-oxo-l-proline, suggesting that the latter compound might interfere with the trans-4-hydroxy-l-proline breakdown in human cells. We conclude that BDH2 is a mammalian 4-oxo-l-proline reductase that converts 4-oxo-l-proline to cis-4-hydroxy-l-proline and not to trans-4-hydroxy-l-proline, as originally thought. We also hypothesize that this enzyme may be a potential source of cis-4-hydroxy-l-proline in mammalian tissues.

The Structure, Activity, and Function of the SETD3 Protein Histidine Methyltransferase.

SETD3 has been recently identified as a long sought, actin specific histidine methyltransferase that catalyzes the Nτ-methylation reaction of histidine 73 (H73) residue in human actin or its equivalent in other metazoans. Its homologs are widespread among multicellular eukaryotes and expressed in most mammalian tissues. SETD3 consists of a catalytic SET domain responsible for transferring the methyl group from S-adenosyl-L-methionine (AdoMet) to a protein substrate and a RuBisCO LSMT domain that recognizes and binds the methyl-accepting protein(s). The enzyme was initially identified as a methyltransferase that catalyzes the modification of histone H3 at K4 and K36 residues, but later studies revealed that the only bona fide substrate of SETD3 is H73, in the actin protein. The methylation of actin at H73 contributes to maintaining cytoskeleton integrity, which remains the only well characterized biological effect of SETD3. However, the discovery of numerous novel methyltransferase interactors suggests that SETD3 may regulate various biological processes, including cell cycle and apoptosis, carcinogenesis, response to hypoxic conditions, and enterovirus pathogenesis. This review summarizes the current advances in research on the SETD3 protein, its biological importance, and role in various diseases.

DHEA-pretreatment attenuates oxidative stress in kidney-cortex and liver of diabetic rabbits and delays development of the disease.

In view of reported discrepancies concerning antioxidant activity of dehydroepiandrosterone (DHEA), a widely used dietary supplement, the current investigation was undertaken to evaluate the antioxidant properties of DHEA in both kidney-cortex and liver of alloxan (ALX)-induced diabetic rabbits, as this diabetogenic compound exhibits the ROS-dependent action. ALX was injected to animals following 7 days of DHEA administration. Four groups of rabbits were used in the experiments: control, DHEA-treated control, diabetic and DHEA-treated diabetic. Our results show for the first time, that in kidney-cortex DHEA resulted in normalization of hydroxyl free radicals (HFR) levels and restoration of catalase (CAT) and glutathione peroxidase (GPx) activities to near the control values, while in liver DHEA prevented the malondialdehyde (MDA) accumulation and normalized glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G6PDH) activities. Moreover, in both kidney-cortex and liver DHEA supplementation prevented GSSG elevation accompanied by a decrease in GSH/GSSG ratio. Although DHEA attenuated oxidative stress in both kidney-cortex and liver of ALX-induced diabetic rabbits and significantly delayed the onset of diabetes in time, it did not protect against the final development of diabetes. In conclusion, the current investigation underscores the complexity of the antioxidant action of DHEA. The data are of clinical interest since DHEA supplementation could prevent the deleterious effects of ROS and delay, or even prevent the onset of many diseases. However, in view of the reported pro-oxidant effects of high DHEA doses, the potential use of this agent as a supplement needs a careful evaluation.