SMUG2 DNA glycosylase from Pedobacter heparinus as a new subfamily of the UDG superfamily.

Base deamination is a common type of DNA damage that occurs in all organisms. DNA repair mechanisms are essential to maintain genome integrity, in which the base excision repair (BER) pathway plays a major role in the removal of base damage. In the BER pathway, the uracil DNA glycosylase superfamily is responsible for excising the deaminated bases from DNA and generates apurinic/apyrimidinic (AP) sites. Using bioinformatics tools, we identified a family 3 SMUG1-like DNA glycoyslase from Pedobacter heparinus (named Phe SMUG2), which displays catalytic activities towards DNA containing uracil or hypoxanthine/xanthine. Phylogenetic analyses show that SMUG2 enzymes are closely related to family 3 SMUG1s but belong to a distinct branch of the family. The high-resolution crystal structure of the apoenzyme reveals that the general fold of Phe SMUG2 resembles SMUG1s, yet with several distinct local structural differences. Mutational studies, coupled with structural modeling, identified several important amino acid residues for glycosylase activity. Substitution of G65 with a tyrosine results in loss of all glycosylase activity. The crystal structure of the G65Y mutant suggests a potential misalignment at the active site due to the mutation. The relationship between the new subfamily and other families in the UDG superfamily is discussed. The present study provides new mechanistic insight into the molecular mechanism of the UDG superfamily.

Structures of a glucose-tolerant ß-glucosidase provide insights into its mechanism.

Cellulose can be converted to ethanol via the fermentation of glucose, which is considered as a promising green alternative for transportation fuels. The conversion of cellulose to glucose needs three enzymes, in which ß-glucosidase (BGL) plays an essential role. However, BGL is inhibited by its own product glucose, greatly limiting its applications in industry. We previously obtained a novel BGL named Bgl6 with a high glucose tolerance. Further engineering through random mutagenesis produced a triple mutant M3 with improved thermostability. This enzyme shows promising properties for wide applications but the structural basis of the unusual properties of Bgl6 is not clear. In this study, we determined the crystal structures of Bgl6 and variants at high resolution, which provide insights into its glucose-tolerant mechanism and thermostability. Particularly, Bgl6 forms an extra channel that could be used as a secondary binding site for glucose, which may contribute to glucose tolerance. Additionally, the triple mutations could strengthen the hydrophobic interactions within the enzyme and may be responsible for the enhanced thermostability exhibited by M3, which was further confirmed by dynamic light scattering data. Lastly, structural comparison to other orthologs allows us to formulate new strategies on how to improve the catalytic efficiency of Bgl6.

Structural and biochemical insights into the 2'-O-methylation of pyrimidines 34 in tRNA.

tRNA molecules undergo extensive modifications during their maturation and these modifications play important cellular roles. TrmL is a tRNA-modification enzyme that catalyzes the transfer of a methyl group to the 2'-hydroxyl group of the pyrimidines at the wobble position 34 in two tRNALeu isoacceptors, but the mechanism remains elusive. In this study, we determined the crystal structure of TrmL from Thermus thermophilus (TtTrmL) to 1.7 Å. The enzyme contains only the conserved minimal SPOUT fold, but displays distinct biochemical behavior from its Escherichia coli counterpart, EcTrmL. Interestingly, a fortuitous ligand of 5'-methylthioadenosine was consistently found at the SAM-binding pocket in the crystal structures, which probably came from the expression host. Both TtTrmL and EcTrmL were capable of methylating each other's tRNA substrates, but the latter exhibited much higher activity than the former. Enzymatic activity assays showed that the reaction catalyzed by TtTrmL greatly depends on the reaction pH and is also affected by salt concentration. Via sequence alignment and structural superposition, we discovered that a universally conserved glutamate residue is likely to fulfill the role of the general base for the initial proton abstraction from the 2'-hydroxyl group of pyrimidines 34. Lastly, based on our structural and biochemical data, we proposed the dimer interface to be the tRNA-binding site for TtTrmL. DATABASE: The atomic coordinates and structural factors have been deposited in the Protein Data Bank with accession number 5CO4.