Brief Communications in 2012

Structural characterization of an artificial zinc-dependent enzyme created by in vitro evolution yields a new, flexible fold that challenges straightforward definitions of active site residues and raises questions about protein evolution.

Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) is a proven methodology for in vitro RNA secondary structure analysis. The identification of a new acylating agent permits the use of SHAPE to probe folded RNAs within living cells.

Biotechnological applications of hydrogenases are limited by their susceptibility to inactivation by oxygen, thought to proceed by trapping a reduced O₂ in the active site. Electrochemical and spectroscopic studies using various electron acceptors now show that oxygen inactivation is not linked to oxygen atom donation.

Understanding the reaction mechanism of OGT, responsible for O-GlcNAcylating various protein substrates, has been hampered by a lack of structural information. Snapshots of ternary complexes along the reaction coordinate now provide evidence for substrate participation in an electrophilic migration mechanism.

The identification of Escherichia coli ycfD and human MINA53 and NO66 as ribosomal amino acid hydroxylases defines a role for 2-oxoglutarate/iron-dependent oxygenases in translational regulation.

Riboswitches—RNA motifs that regulate gene expression in response to binding of specific ligands—have been identified for many small-molecule metabolites. An ATP-binding element in the ydaO mRNA of Bacillus subtilis provides the first example of an ATP-responsive riboswitch.

TsrM, a member of the radical SAM enzyme family, is shown to catalyze tryptophan methylation en route to thiostrepton A with the help of a methylcobalamin cofactor and without generating the canonical 5-deoxyadenosyl radical.

A new approach for rational enzyme design uses gain-of-function cancer mutations to guide homologous mutations in homoisocitrate dehydrogenase, yielding a biocatalytic path to (R)-2-hydroxyadipate, a precursor for the major commodity chemical adipic acid.

The mechanism for nitro group formation in the thaxtomin family of natural products is unknown. Genetic and biochemical studies now show the cytochrome P450 TxtE catalyzes this direct and regioselective nitration, using NO and O₂ to modify a tryptophan indole ring.

Genetic code expansion by ribosomal incorporation of non-natural amino acids has provided a useful approach for site-specific protein modification. This approach has now been extended to the model organism Drosophila melanogaster, permitting the introduction of non-standard amino acids into proteins within specific cell and tissue types and across developmental stages.

AWP28, an activity-based probe for caspase-1, reveals that caspase-1 is required to bypass apoptosis for pyroptotic cell death during bacterial infection of macrophages.

Bacterial resistance is propagated in part by metallo-β-lactamases, which hydrolyze and inactivate β-lactam antibiotics. An unusual cysteine residue in the active site is now shown to be critical for retaining the second metal ion, and thus enzyme activity, at low zinc concentrations.

Elongation factor P is a conserved translational regulatory protein that has an unusual post-translational modification, in which Lys34 forms an amide linkage to (R)-β-lysine. Further characterization reveals that Lys34 is also hydroxylated, drawing parallels to a functional modification of eukaryotic initiation factor 5A.

Many efforts to expand the genetic alphabet and reprogram the genetic code have relied on synthetic DNA nucleotides designed to have pairing properties orthogonal to those of natural base pairs. A structural study shows that DNA polymerases enhance the efficiency of non-natural base pair replication by enforcing a standard Watson-Crick geometry in the polymerase active site.

Mapping of a mutation in a tomato deficient in the plant cuticle component cutin yields the first cutin synthase, as shown via accumulation of polymer precursors and in vitro oligomerization of synthetic substrates.

N-linked glycoprotein production requires attachment of eukaryotic glycans to eukaryotic proteins. The introduction of four eukaryotic glycosyltransferases and a bacterial oligosaccharyltransferase now allows E. coli to produce and transfer a five-glycan Man₃GlcNAc₂ eukaryotic core structure to several protein targets.

Searching chemical space for biologically active molecules requires facile access to new molecular architectures. Variations in reagent, catalyst and reaction order create a programmable one-pot method that yields single stereoisomers of complex cycloadducts, including either isomer of enantiomeric pairs.

Indole, secreted by E. coli, induces oxidative-stress and phage-shock pathway genes to increase persistence, a phenomenon in which dormant bacteria are resistant to antibiotics.

The iterative, highly-reducing polyketide synthases use a single copy of each domain to transform multiple substrates, defying conventional rules regarding enzyme function. Synthetic tool compounds and hybrid constructs now provide insights into the specificity of the ketoreductase in dehydrozearalenol biosynthesis.

Selection of clones resistant to drugs in human cells, followed by massively parallel transcriptome sequencing of these clones and bioinformatics analyses to identify genes mutated with high frequency, allow for identification of direct targets and indirect resistance mechanisms of cytotoxic drugs.