Posttranslational modifications of histones regulate chromatin structure and gene expression. Histone demethylases, members of a newly emerging transcription-factor family, remove methyl groups from the lysine residues of the histone tails and thereby regulate the transcriptional activity of target genes. JmjC-domain-containing proteins have been predicted to be demethylases. For example, the JmjC-containing protein JMJD2A has been characterized as a H3-K9me3- and H3-K36me3-specific demethylase. Here, structures of the catalytic-core domain of JMJD2A with and without alpha-ketoglutarate in the presence of Fe2+ have been determined by X-ray crystallography. The structure of the core domain, consisting of the JmjN domain, the JmjC domain, the C-terminal domain, and a zinc-finger motif, revealed the unique elements that form a potential substrate binding pocket. Sited-directed mutagenesis in conjunction with demethylase activity assays allowed us to propose a molecular model for substrate selection by the JMJD2 histone demethylase family.
Recent reports of the slow-tight binding inhibition of bovine liver dihydrofolate reductase (bDHFR) in the presence of polyphenols isolated from green tea leaves has spurred renewed interest in the biochemical properties of bDHFR. Earlier studies were done with native bDHFR but in order to validate models of polyphenol binding to bDHFR, larger quntities of bDHFR are necessary to support structural studies. Bovine DHFR differs from its closest sequence homologue, murine DHFR, by 19 amino acids. To obtain the bDHFR cDNA, murineDHFR cDNA was transformed by a series of nested PCR reactions to reproduce the amino acid coding sequence for bovine DHFR. The bovine liver DHFR cDNA has an open reading frame of 561 base pairs encoding a protein of 187 amino acids that has a high level of conservation at the primary sequence level with other DHFR enzymes, and more so for the amino acid residues in the active site of the mammalian DHFR enzymes. Expression of the bovine DHFR cDNA in bacterial cells produced a stable recombinant protein with high enzymatic activity and kinetic properties similar to those previously reported for the native protein.
The gene product of fabG from Aquifex aeolicus has been heterologously expressed in Escherichia coli. Purification of the protein took place using anionexchange and size-exclusion chromatography and the protein was then crystallized. Diffraction data were collected to a maximum resolution of 1.8 Å and the initial phases were determined by molecular replacement. The A. aeolicus FabG protein is a putative -ketoacyl-acyl carrier protein reductase. Structure-function studies of this protein are being performed as part of a larger project investigating naturally occurring deviations from highly conserved residues within the short-chain oxidoreductase (SCOR) family.
We have identified a highly conserved fingerprint of 40 residues in the TGYK subfamily of the short-chain oxidoreductase enzymes. The TGYK subfamily is defined by the presence of an N-terminal TGxxxGxG motif and a catalytic YxxxK motif. This subfamily contains more than 12,000 members, with individual members displaying unique substrate specificities. The 40 fingerprint residues are critical to catalysis, cofactor binding, protein folding, and oligomerization but are substrate independent. Their conservation provides critical insight into evolution of the folding and function of TGYK enzymes. Substrate specificity is determined by distinct combinations of residues in three flexible loops that make up the substrate-binding pocket. Here, we report the structure determinations of the TGYK enzyme A3DFK9 from Clostridium thermocellum in its apo form and with bound NAD(+) cofactor. The function of this protein is unknown, but our analysis of the substrate-binding loops putatively identifies A3DFK9 as a carbohydrate or polyalcohol metabolizing enzyme. C. thermocellum has potential commercial applications because of its ability to convert biomaterial into ethanol. A3DFK9 contains 31 of the 40 TGYK subfamily fingerprint residues. The most significant variations are the substitution of a cysteine (Cys84) for a highly conserved glycine within a characteristic VNNAG motif, and the substitution of a glycine (Gly106) for a highly conserved asparagine residue at a helical kink. Both of these variations occur at positions typically participating in the formation of a catalytically important proton transfer network. An alternate means of stabilizing this proton wire was observed in the A3DFK9 crystal structures.
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