Biochemical Characterization of an Adenylate Cyclase, CyaB1, in the Cyanobacterium Anabaena sp. Strain PCC 7120

cyaB1 gene encodes a novel type of adenylate cyclase. The catalytic domain is located in the carboxyl-terminal half, while the GAF and PAS domains are conserved in the amino-terminal half. Recombinant CyaB1 and a truncated CyaB1 lacking the amino-terminal domain (ΔN–CyaB1) were purified and characterized. The purified CyaB1 is activated by divalent cations, such as Mg²⁺ and Mn²⁺, like other types of adenylate cyclase. The activity of CyaB1 was slightly elevated by forskolin, but was not affected by cGMP, irrespective of the presence of the cGMP binding motif in the GAF domain. The specific activity of ΔN–CyaB1 is one-eighteenth that of CyaB1, whereas the Km values of both proteins are almost the same. The results suggest that the amino-terminal half has a positive regulatory effect on the catalytic activity. Received 27 April 2001/ Accepted in revised form 6 July 2001     

Small-angle X-ray scattering reveals the solution structure of a bacteriophytochrome in the catalytically active Pr state

Phytochromes are light-sensing macromolecules that are part of a two component phosphorelay system controlling gene expression. Photoconversion between the Pr and Pfr forms facilitates autophosphorylation of a histidine in the dimerization domain (DHp). We report the low-resolution structure of a bacteriophytochrome (Bph) in the catalytic (CA) Pr form in solution determined by small-angle X-ray scattering (SAXS). Ab initio modeling reveals, for the first time, the domain organization in a typical bacteriophytochrome, comprising an chromophore binding and phytochrome (PHY) N terminal domain followed by a C terminal histidine kinase domain. Homologous high-resolution structures of the light-sensing chromophore binding domain (CBD) and the cytoplasmic part of a histidine kinase sensor allows us to model 75% of the structure with the remainder comprising the phytochrome domain which has no 3D representative in the structural database. The SAXS data reveal a dimeric Y shaped macromolecule and the relative positions of the chromophores (biliverdin), autophosphorylating histidine residues and the ATP molecules in the kinase domain. SAXS data were collected from a sample in the autophosphorylating Pr form and reveal alternate conformational states for the kinase domain that can be modeled in an open (no-catalytic) and closed (catalytic) state. This model suggests how light-induced signal transduction can stimulate autophosphorylation followed by phosphotransfer to a response regulator (RR) in the two-component system.     

Conformational conversion of PDE5 by incubation with sildenafil or metal ion is accompanied by stimulation of allosteric cGMP binding

Phosphodiesterase-5 (PDE5) is a dimer containing a cGMP-specific catalytic domain and an allosteric cGMP-binding subdomain (GAF A) on each subunit. PDE5 exhibits three conformational forms that can be separated by Native PAGE and are denoted as Bands 1, 2, and 3 in decreasing order of mobility. A preparation comprised mainly of Band 2 PDE5 was partially converted to Band 3 PDE5 by 1 h incubation with cGMP or the PDE5-specific inhibitors sildenafil, vardenafil, or tadalafil, but not with cAMP, milrinone (PDE3-specific), or rolipram (PDE4-specific). Band 2 PDE5 was converted almost entirely to Band 3 PDE5 by overnight incubation with sildenafil at 30 °C. This time-dependent conversion was accompanied by a 7-fold increase in allosteric cGMP-binding activity, suggesting that Band 3 PDE5 is a much more active form than Band 2 PDE5 for allosteric cGMP binding. Conversion of Band 2 PDE5 to Band 3 PDE5 occurred faster by pre-incubation with cGMP, which binds to both the allosteric and catalytic sites of PDE5, than with catalytic site-specific sildenafil. Overnight incubation of a Band 2/Band 3 PDE5 mixture with EDTA caused time-dependent conversion to Band 1 PDE5 (apoenzyme), and this conversion was accompanied by a 50% loss in cGMP-binding activity. After incubation with EDTA, addition of Mn⁺⁺ or Mg⁺⁺ caused reversion of Band 1 to a Band 2/Band 3 PDE5 mixture in which Band 3 PDE5 predominated. This reversion was accompanied by a 3-fold increase in allosteric cGMP-binding activity. The combination of results implied that physiological conversion of Band 2 to Band 3 PDE5 by cGMP and/or divalent metal ion occupancy of the catalytic domain would increase allosteric cGMP binding to the enzyme. This conversion would produce a greater negative feedback effect on cGMP action by increasing sequestration of cGMP at the allosteric cGMP-binding site of PDE5 and by increasing cGMP degradation at the catalytic site of the enzyme. This conversion would also increase PDE5 inhibitor binding to the enzyme.     

Metal ion stimulators of PDE5 cause similar conformational changes in the enzyme as does cGMP or sildenafil

Purified PDE5 preparations exhibited variable proportions of two mobility forms (Bands 2 and 3) by native PAGE. Treatment of recombinant or native PDE5 with either cGMP or a substrate analog such as sildenafil, each of which is known to produce stimulatory effects on enzyme functions, caused a similar native PAGE band-shift to the lower mobility form (shift of Band 2 to Band 3). Incubation of PDE5 with Mg⁺⁺ or Mn⁺⁺, which is known to stimulate activity, caused a similar shift of the enzyme from Band 2 to Band 3 as did cGMP or sildenafil, but incubation with EDTA caused a time- and concentration-dependent shift to higher mobility (shift of Bands 2 and 3 to Band 1). A slow time course of the EDTA-induced band-shift suggested removal of a pre-bound metal ion (Me⁺⁺) with affinity of ~ 0.1 nM, which was similar to the previously determined affinity of PDE5 for Zn⁺⁺. The EDTA-treated enzyme (Band 1) could be shifted to Bands 2 and 3 by addition of cGMP, sildenafil, or Me⁺⁺; however, the cGMP- or sildenafil-induced shift was inhibited and the Me⁺⁺-induced shift was facilitated by treatment with EDTA. Results suggested that Me⁺⁺ removal from PDE5 produces a unique apoenzyme form (Band 1, more globular, negatively charged, or both) of PDE5 that can be partially converted to forms (Band 2, less globular or negatively charged, or both; and Band 3, more elongated/positively charged, or both) by addition of Me⁺⁺, substrate, or substrate analog. It is concluded that Me⁺⁺ causes conversion of PDE5 to similar conformational forms as caused by substrate or inhibitor binding to the catalytic site.     

Surviving an identity crisis: A revised view of chromatin insulators in the genomics era

The control of complex, developmentally regulated loci and partitioning of the genome into active and silent domains is in part accomplished through the activity of DNA-protein complexes termed chromatin insulators. Together, the multiple, well-studied classes of insulators in Drosophila melanogaster appear to be generally functionally conserved. In this review, we discuss recent genomic-scale experiments and attempt to reconcile these newer findings in the context of previously defined insulator characteristics based on classical genetic analyses and transgenic approaches. Finally, we discuss the emerging understanding of mechanisms of chromatin insulator regulation. This article is part of a Special Issue entitled: Chromatin and epigenetic regulation of animal development.     

A novel photoactive GAF domain of cyanobacteriochrome AnPixJ that shows reversible green/red photoconversion

We report the discovery of a novel cyanobacteriochrome, the green/red photoreceptor AnPixJ (All1069), isolated from the heterocyst-forming cyanobacterium Anabaena (Nostoc) sp. PCC 7120. Cyanobacteriochromes are a recently emerging tetrapyrrole-based photoreceptor superfamily that are distantly related to the conventional red/far-red photoreceptor phytochromes (Phys). The chromophore-binding domains of AnPixJ produced in cyanobacterial and Escherichia coli cells both showed a reversible and full photoconversion between a green-absorbing form (λ_max = 543 nm) and a red-absorbing form (λ_max = 648 nm). Denaturation analysis revealed that the green-absorbing form and the red-absorbing form covalently ligated phycocyanobilin with E-configuration and Z-configuration at the C15C16 double bond, respectively. Time-resolved spectral analysis showed the formation of the first intermediate state peaking at 680 nm from the dark-stable red-absorbing form. This step resembles the first photoconversion step from the red-absorbing form to the red-shifted lumi-R intermediate state of the Phys. These results suggest that the Pr of AnPixJ is almost equivalent to that of the Phys and starts a primary photoreaction with Z-to-E isomerization in a mechanism similar to that in the Phys, but is finally photoconverted to the unique green-absorbing form.