No abstract

Biological sulfide oxidation is a reaction occurring in all three domains of life. One enzyme responsible for this reaction in many bacteria has been identified as sulfide:quinone oxidoreductase (SQR). The enzyme from Rhodobacter capsulatus is a peripherally membrane-bound flavoprotein with a molecular mass of approximately 48 kDa, presumably acting as a homodimer. In this work, SQR from Rb. capsulatus has been modified with an N-terminal His tag and heterologously expressed in and purified from Escherichia coli. Three cysteine residues have been shown to be essential for the reductive half-reaction by site-directed mutagenesis. The catalytic activity has been nearly completely abolished after mutation of each of the cysteines to serine. A decrease in fluorescence on reduction by sulfide as observed for the wild-type enzyme has not been observed for any of the mutated enzymes. Mutation of a conserved valine residue to aspartate within the third flavin-binding domain led to a drastically reduced substrate affinity, for both sulfide and quinone. Two conserved histidine residues have been mutated individually to alanine. Both of the resulting enzymes exhibited a shift in the pH dependence of the SQR reaction. Polysulfide has been identified as a primary reaction product using spectroscopic and chromatographic methods. On the basis of these data, reaction mechanisms for sulfide-dependent reduction and quinone-dependent oxidation of the enzyme and for the formation of polysulfide are proposed.

It is generally accepted that the endothelium regulates vascular tone independent of the activity of the sympathetic nervous system. Here, we tested the hypothesis that the activation of sympathetic nerves engages the endothelium to oppose vasoconstriction. Local inositol 1,4,5-trisphosphate (IP(3))-mediated Ca(2+) signals ("pulsars") in or near endothelial projections to vascular smooth muscle (VSM) were measured in an en face mouse mesenteric artery preparation. Electrical field stimulation of sympathetic nerves induced an increase in endothelial cell (EC) Ca(2+) pulsars, recruiting new pulsar sites without affecting activity at existing sites. This increase in Ca(2+) pulsars was blocked by bath application of the α-adrenergic receptor antagonist prazosin or by TTX but was unaffected by directly picospritzing the α-adrenergic receptor agonist phenylephrine onto the vascular endothelium, indicating that nerve-derived norepinephrine acted through α-adrenergic receptors on smooth muscle cells. Moreover, EC Ca(2+) signaling was not blocked by inhibitors of purinergic receptors, ryanodine receptors, or voltage-dependent Ca(2+) channels, suggesting a role for IP(3), rather than Ca(2+), in VSM-to-endothelium communication. Block of intermediate-conductance Ca(2+)-sensitive K(+) channels, which have been shown to colocalize with IP(3) receptors in endothelial projections to VSM, enhanced nerve-evoked constriction. Collectively, our results support the concept of a transcellular negative feedback module whereby sympathetic nerve stimulation elevates EC Ca(2+) signals to oppose vasoconstriction.

The type I cGMP-dependent protein kinases serve essential physiological functions, including smooth muscle relaxation, cardiac remodeling, and platelet aggregation. These enzymes form homodimers through their N-terminal dimerization domains, a feature implicated in regulating their cooperative activation. Previous investigations into the activation mechanisms of PKG I isoforms have been largely influenced by structures of the cAMPdependent protein kinase (PKA). Here, we examined PKG Ia activation by cGMP and cAMP by engineering a monomeric form that lacks N-terminal residues 1-53 ( 53). We found that the construct exists as a monomer as assessed by whole-protein mass spectrometry, size-exclusion chromatography, and small-angle X-ray scattering (SAXS). Reconstruction of the SAXS 3D envelope indicates that 53 has a similar shape to the heterodimeric RIa:C complex of PKA. Moreover, we found that the 53 construct is autoinhibited in its cGMP-free state and can bind to and be activated by cGMP in a manner similar to full-length as assessed by surface plasmon resonance spectroscopy (SPR). However we found that the 53 variant does not exhibit cooperative activation, and its cyclic nucleotide selectivity is diminished. These findings support a model in which, despite structural similarities, PKG Ia activation is distinct from PKA, and its cooperativity is driven by in trans interactions between protomers.

In eukaryotes, the poly(A) tail added at the 3′ end of a mRNA precursor is essential for the regulation of mRNA stability and the initiation of translation. Poly(A) polymerase (PAP) is the enzyme that catalyzes the poly(A) addition reaction. Multiple isoforms of PAP have been identified in vertebrates, which originate from gene duplication, alternative splicing, or post-translational modifications. The complexity of PAP isoforms suggests that they might play different roles in the cell. Phylogenetic studies indicate that vertebrate PAPs are grouped into three clades termed α, β and γ, which originated from two gene duplication events. To date, all the available PAP structures are from the PAPα clade. Here, we present the crystal structure of the first representative of the PAPγ clade, human PAPγ, bound to cordycepin triphosphate (3′dATP) and Ca2+. The structure revealed that PAPγ closely resembles its PAPα ortholog. An analysis of residue conservation reveals a conserved catalytic binding pocket, whereas residues at the surface of the polymerase are more divergent.