Biology subjects

Drozak, J.

Publications and source records attributed to Drozak, J..

4 recordsLinked to original sources

bioRxiv2021

Substrate Specificity for Human Histidine Methyltransferase SETD3

Histidine methyltransferase SETD3 plays an important role in human biology and diseases. Previously, we showed that SETD3 catalyzes N3-methylation of histidine 73 in {beta}-actin (Kwiatkowski et al., 2018). Here we report integrated synthetic, biocatalytic, biostructural and computational analyses on human SETD3-catalyzed methylation of {beta}-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 (QM/MM) 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 {beta}-actin histidine methylation by SETD3.

biochemistry↗

bioRxiv2021

The Mammalian Cytosolic Type 2 (R)-β-hydroxybutyrate Dehydrogenase (BDH2) is 4-oxo-L-proline Reductase (EC1.1.1.104)

The early studies on chicken embryos revealed that exposition to 4-oxo-L-proline resulted in the explicit increase in 4-hydroxy-L-proline content in their tissues. 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, but only recently the molecular identity of the enzyme has been unveiled in our laboratory. The present investigation reports the purification, identification as well as biochemical characterization of 4-oxo-L-proline reductase. The enzyme was purified from rat kidneys about 280-fold. Following mass spectrometry analysis of the purified protein preparation, the mammalian cytosolic type 2 (R)-{beta}-hydroxybutyrate dehydrogenase (BDH2) emerged as the only meaningful candidate for the reductase. Rat and human BDH2 were expressed in E. coli, purified, and shown to catalyze the reversible reduction of 4-oxo-L-proline to cis-4-hydroxy-L-proline, as confirmed by chromatographic and mass spectrometry analysis. Specificity studies carried out on both enzymes showed that 4-oxo-L-proline was the best substrate, particularly the human enzyme acted with 9400-fold higher catalytic efficiencies on 4-oxo-L-proline than on (R)-{beta}-hydroxybutyrate. Finally, HEK293T cells efficiently metabolized 4-oxo-L-proline to cis-4-hydroxy-L-proline and simultaneously accumulated trans-4-hydroxy-L-proline in the culture medium, suggesting that 4-oxo-L-proline is most likely an inhibitor of trans-4-hydroxy-L-proline metabolism in human cells. We conclude that BDH2 is 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 currently thought, and hypothesize that the enzyme may be considered as a potential source of cis-4-hydroxy-L-proline in mammalian tissues.

biochemistry↗

bioRxiv2018

Structural insights into SETD3-mediated histidine methylation on β-actin

SETD3 is a member of SET (Su(var)3-9, Enhancer of zeste, and Trithorax) domain protein superfamily and plays important roles in hypoxic pulmonary hypertension, muscle differentiation, and carcinogenesis. In a previous paper (Kwiatkowski et al. 2018), we have identified SETD3 as the actin-specific methyltransferase that methylates the N3 of His73 on {beta}-actin. Here we present two structures of S-adenosyl-L-homocysteine-bound SETD3 in complex with either an unmodified {beta}-actin peptide or its His-methylated variant. Structural analyses supported by the site-directed mutagenesis experiments and the enzyme activity assays indicated that the recognition and methylation of {beta}-actin by SETD3 is highly sequence specific, and both SETD3 and {beta}-actin adopt pronounce conformational changes upon binding to each other. In conclusion, the structural research uncovers the molecular mechanism of sequence-selective histidine methylation by SETD3, which not only throws light on protein histidine methylation phenomenon, but also facilitates the design of small molecule inhibitors of SETD3.

biochemistry↗

bioRxiv2018

SETD3 protein is the actin-specific histidine N-methyltransferase

Protein histidine methylation is rarely studied posttranslational modification of unknown biochemical importance. In vertebrates, only a few methylhistidne-containing proteins have been reported so far, including {beta}-actin as an essential example. The evolutionary conserved methylation of {beta}-actin H73 residue is catalyzed by a specific histidine N-methyltransferase that has never been identified molecularly. In the present investigation, we have purified actin-specific histidine N-methyltransferase from rat muscles about 1200-fold. Its activity was studied by the radiochemical assay employing either homogeneous recombinant human {beta}-actin produced in E. coli or its mutated form exhibiting substitution of H73 by Ala residue (H73A) as substrates. Three polypeptides of {approx}65, 75 and 90 kDa coeluting with the enzyme activity were identified in the preparation. Mass spectrometry analysis of these polypeptides resulted in the identification of SETD3 methyltransferase as the only plausible candidate. Rat SETD3 and its human ortholog were expressed in COS-7 cells, purified to homogeneity and shown to catalyze methylation of {beta}-actin at H73 residue as confirmed by mass spectrometry analysis. The SETD3 enzyme was active towards a synthetic peptide corresponding to residues 69-77 of {beta}-actin, but not to its mutated form exhibiting His-to-Ala substitution. Finally, Setd3-deficient HAP1 cells were devoid of methylated H73 in {beta}-actin and exhibited phenotypic changes, including a decrease in F-actin content and an increased glycolytic activity. We conclude that SETD3 is the actin-specific histidine N-methyltransferase. The data show for the first time the molecular identity of protein histidine N-methyltransferase in vertebrates and throw new light on the substrate specificity of SET-domain-containing enzymes.

biochemistry↗