A [3Fe-4S] cluster and tRNA-dependent aminoacyltransferase BlsK in the biosynthesis of Blasticidin S

Blasticidin S is a peptidyl nucleoside antibiotic. Its biosynthesis involves a cryptic leucylation and two leucylated intermediates, LDBS and LBS, have been found in previous studies. Leucylation has been proposed to be a new self-resistance mechanism during blasticidin S biosynthesis, and the leucyl group was found to be important for the methylation of β-amino group of the arginine side chain. However, the responsible enzyme and its associated mechanism of the leucyl transfer process remain to be elucidated. Here, we report results investigating the leucyl transfer step forming the intermediate LDBS in blasticidin biosynthesis. A hypothetical protein, BlsK, has been characterized by genetic and in vitro biochemical experiments. This enzyme catalyzes the leucyl transfer from leucyl-transfer RNA (leucyl-tRNA) to the β-amino group on the arginine side chain of DBS. Furthermore, BlsK was found to contain an iron–sulfur cluster that is necessary for activity. These findings provide an example of an iron–sulfur protein that catalyzes an aminoacyl-tRNA (aa-tRNA)–dependent amide bond formation in a natural product biosynthetic pathway.

Polyunsaturated fatty acid structure determines the strength of potentiation of Acid-sensing ion channels

Acid-sensing ion channels (ASICs) are thought to be endogenous sensors of acidic pain in inflammatory pathways. It has previously been demonstrated that arachidonic acid (AA), a pain and inflammation promoting molecule, potentiates ASICs. However, a mechanistic understanding of how AA regulates ASICs is lacking. Furthermore, little is known regarding modulation by other polyunsaturated fatty acids (PUFAs). Here we show that PUFAs stabilize the open state of the channel by shifting the pH dependence of activation to more alkaline values, increasing max conductance, and slowing channel desensitization. We examine the effects of 35 PUFAs/PUFA derivatives and show that ASICs can be more strongly potentiated by these lipids than was originally seen for AA. In fact, arachidonoyl glycine (AG) can act as a ligand and activate the channel in the absence of acidic pH. We find that the strength of potentiation is critically dependent upon a negatively charged PUFA head group as well as both the length and the number of doubles bonds in the acyl tail. PUFA-induced shifts in the pH dependence of activation could be eliminated upon mutation of a highly conserved, positively charged arginine in the outer segment of TM1 (R64). Combined our results suggest a hypothesis whereby an electrostatic interaction between the charged PUFA head group and the positively charged arginine side chain potentiates ASIC currents by stabilizing the open state of the channel. This work uncovers a novel putative lipid binding site on ASICs and provides the structural basis for future development of compounds targeting ASICs.

Methylguanidinium at the Air/Water Interface: A Simulation Study with the Drude Polarizable Force Field

<div>Methylguanidinium is an important molecular ion which also serves as the model compound for arginine side chain. We studied the structure and dynamics of methylguanidium ion at the air/water interface by molecular dynamics simulations employing the Drude polarizable force field. We found out that methylguanidinium accumulate on the interface with a majority adopting tilted conformations. We also demonstrated that methylguanidinium and guanidinium ions have different preference towards the air/water interface. Our results illustrate the importance to explicitly include the electronic polarization effects in modeling interfacial properties.</div><div><br> </div>

Methylguanidinium at the Air/Water Interface: A Simulation Study with the Drude Polarizable Force Field

<div>Methylguanidinium is an important molecular ion which also serves as the model compound for arginine side chain. We studied the structure and dynamics of methylguanidium ion at the air/water interface by molecular dynamics simulations employing the Drude polarizable force field. We found out that methylguanidinium accumulate on the interface with a majority adopting tilted conformations. We also demonstrated that methylguanidinium and guanidinium ions have different preference towards the air/water interface. Our results illustrate the importance to explicitly include the electronic polarization effects in modeling interfacial properties.</div><div><br> </div>

Arginine off-kilter: guanidinium is not as planar as restraints denote

Crystallographic refinement of macromolecular structures relies on stereochemical restraints to mitigate the typically poor data-to-parameter ratio. For proteins, each amino acid has a unique set of geometry restraints which represent stereochemical information such as bond lengths, valence angles, torsion angles, dihedrals and planes. It has been shown that the geometry in refined structures can differ significantly from that present in libraries; for example, it was recently reported that the guanidinium moiety in arginine is not symmetric. In this work, the asymmetry of the N∊—Cζ—Nη1 and N∊—Cζ—Nη2 valence angles in the guanidinium moiety is confirmed. In addition, it was found that the Cδ atom can deviate significantly (more than 20°) from the guanidinium plane. This requires the relaxation of the planar restraint for the Cδ atom, as it otherwise causes the other atoms in the group to compensate by distorting the guanidinium core plane. A new set of restraints for the arginine side chain have therefore been formulated, and are available in the software package Phenix, that take into account the asymmetry of the group and the planar deviation of the Cδ atom. This is an example of the need to regularly revisit the geometric restraint libraries used in macromolecular refinement so that they reflect the best knowledge of the structural chemistry of their components available at the time.

Visualizing Rev1 catalyze protein-template DNA synthesis

During DNA replication, replicative DNA polymerases may encounter DNA lesions, which can stall replication forks. One way to prevent replication fork stalling is through the recruitment of specialized translesion synthesis (TLS) polymerases that have evolved to incorporate nucleotides opposite DNA lesions. Rev1 is a specialized TLS polymerase that bypasses abasic sites, as well as minor-groove and exocyclic guanine adducts. Lesion bypass is accomplished using a unique protein-template mechanism in which the templating base is evicted from the DNA helix and the incoming dCTP hydrogen bonds with an arginine side chain of Rev1. To understand the protein-template mechanism at an atomic level, we employed a combination of time-lapse X-ray crystallography, molecular dynamics simulations, and DNA enzymology on theSaccharomyces cerevisiaeRev1 protein. We find that Rev1 evicts the templating base from the DNA helix prior to binding the incoming nucleotide. Binding the incoming nucleotide changes the conformation of the DNA substrate to orient it for nucleotidyl transfer, although this is not coupled to large structural changes in Rev1 like those observed with other DNA polymerases. Moreover, we found that following nucleotide incorporation, Rev1 converts the pyrophosphate product to two monophosphates, which drives the reaction in the forward direction and prevents pyrophosphorolysis. Following nucleotide incorporation, the hydrogen bonds between the incorporated nucleotide and the arginine side chain are broken, but the templating base remains extrahelical. These postcatalytic changes prevent potentially mutagenic processive synthesis by Rev1 and facilitate dissociation of the DNA product from the enzyme.

Methylguanidinium at the Air/Water Interface: A Simulation Study with the Drude Polarizable Force Field

<div>Methylguanidinium is an important molecular ion which also serves as the model compound for arginine side chain. We studied the structure and dynamics of methylguanidium ion at the air/water interface by molecular dynamics simulations employing the Drude polarizable force field. We found out that methylguanidinium accumulate on the interface with a majority adopting tilted conformations. We also demonstrated that methylguanidinium and guanidinium ions have different preference towards the air/water interface. Our results illustrate the importance to explicitly include the electronic polarization effects in modeling interfacial properties.</div><div><br> </div>

Visualizing Rev1 Catalyze Protein-template DNA Synthesis

During DNA replication, replicative DNA polymerases may encounter DNA lesions, which can stall replication forks. One way to prevent replication fork stalling is through the recruitment of specialized translesion synthesis (TLS) polymerases that have evolved to incorporate nucleotides opposite DNA lesions. Rev1 is a specialized TLS polymerase that bypasses abasic sites as well as minor-groove and exocyclic guanine adducts. It does this by using a unique protein-template mechanism in which the template base is flipped out of the DNA helix and the incoming dCTP hydrogen bonds with an arginine side chain. To observe Rev1 catalysis at the atomic level, we employed time-lapse X-ray crystallography. We found that Rev1 flips out the template base prior to binding the incoming nucleotide. Binding the incoming nucleotide changes the conformation of the DNA substrate to orient it for nucleotidyl transfer, and this is not coupled to large structural changes in the protein like those observed with other DNA polymerases. Moreover, we found that following nucleotide incorporation, Rev1 converts the pyrophosphate product to two mono-phosphates, which drives the reaction in the forward direction. Following nucleotide incorporation, the hydrogen bonds between the incorporated nucleotide and the arginine side chain are broken, but the templating base remains extrahelical. These post-catalytic changes prevent potentially mutagenic processive synthesis by Rev1 and facilitate dissociation of the DNA product from the enzyme.