The integral membrane protein porin from Rhodobacter capsulatus consists of three tightly associated 16-stranded beta barrels that give rise to three distinct diffusion channels for small solutes through the outer membrane. The x-ray structure of this porin has revealed details of its shape, the residue distributions within the pore and at the membrane-facing surface, and the location of calcium sites. The electrostatic potential has been calculated and related to function. Moreover, potential calculations were found to predict the Ca2+ sites.

SummaryToluene is anoxically degraded to CO 2 by the denitrifying bacterium Thauera aromatica. The initial reaction in this pathway is the addition of fumarate to the methyl group of toluene, yielding benzylsuccinate as the first intermediate. We purified the enzyme catalysing this reaction, benzylsuccinate synthase (EC 4.1.99-), and studied its properties. The enzyme was highly oxygen sensitive and contained a redox-active flavin cofactor, but no iron centres. The native molecular mass was 220 kDa; four subunits of 94 (␣), 90 (␣Ј), 12 (␤) and 10 kDa (␥) were detected on sodium dodecyl sulphate (SDS) gels. The N-terminal sequences of the ␣-and ␣Ј-subunits were identical, suggesting a C-terminal degradation of half of the ␣-subunits to give the ␣Ј-subunit. The composition of native enzyme therefore appears to be ␣ 2 ␤ 2 ␥ 2 . A 5 kb segment of DNA containing the genes for the three subunits of benzylsuccinate synthase was cloned and sequenced. The masses of the predicted gene products correlated exactly with those of the subunits, as determined by electrospray mass spectrometry. Analysis of the derived amino acid sequences revealed that the large subunit of the enzyme shares homology to glycyl radical enzymes, particularly near the predicted radical site. The highest similarity was observed with pyruvate formate lyases and related proteins. The radical-containing subunit of benzylsuccinate synthase is oxygenolytically cleaved at the site of the glycyl radical, producing the ␣Ј-subunit. The predicted cleavage site was verified using electrospray mass spectrometry. In addition, a gene coding for an activating protein catalysing glycyl radical formation was found. The four genes for benzylsuccinate synthase and the activating enzyme are organized as a single operon; their transcription is induced by toluene. Synthesis of the predicted gene products was achieved in Escherichia coli in a T7-promotor/polymerase system.

The arginine methyltransferase PRMT6 (protein arginine methyltransferase 6) has been shown recently to regulate DNA repair and gene expression. As arginine methylation of histones is an important mechanism in transcriptional regulation, we asked whether PRMT6 possesses activity toward histones. We show here that PRMT6 methylates histone H3 at R2 and histones H4/H2A at R3 in vitro. Overexpression and knockdown analysis identify PRMT6 as the major H3 R2 methyltransferase in vivo. We find that H3 R2 methylation inhibits H3 K4 trimethylation and recruitment of WDR5, a subunit of the MLL (mixed lineage leukemia) K4 methyltransferase complex, to histone H3 in vitro. Upon PRMT6 overexpression, transcription of Hox genes and Myc-dependent genes, both well-known targets of H3 K4 trimethylation, decreases. This transcriptional repression coincides with enhanced occurrence of H3 R2 methylation and PRMT6 as well as reduced levels of H3 K4 trimethylation and MLL1/WDR5 recruitment at the HoxA2 gene. Upon retinoic acid-induced transcriptional activation of HoxA2 in a cell model of neuronal differentiation, PRMT6 recruitment and H3 R2 methylation are diminished and H3 K4 trimethylation increases at the gene. Our findings identify PRMT6 as the mammalian methyltransferase for H3 R2 and establish the enzyme as a crucial negative regulator of H3 K4 trimethylation and transcriptional activation.

Homologs of the ferric uptake regulator Fur and the iron storage protein ferritin play a central role in maintaining iron homeostasis in bacteria. The gastric pathogen Helicobacter pylori contains an iron-induced prokaryotic ferritin (Pfr) which has been shown to be involved in protection against metal toxicity and a Fur homolog which has not been functionally characterized in H. pylori. Analysis of an isogenic fur-negative mutant revealed that H. pylori Fur is required for metal-dependent regulation of ferritin. Iron starvation, as well as medium supplementation with nickel, zinc, copper, and manganese at nontoxic concentrations, repressed synthesis of ferritin in the wild-type strain but not in the H. pylori fur mutant. Fur-mediated regulation of ferritin synthesis occurs at the mRNA level. With respect to the regulation of ferritin expression, Fur behaves like a global metal-dependent repressor which is activated under iron-restricted conditions but also responds to different metals. Downregulation of ferritin expression by Fur might secure the availability of free iron in the cytoplasm, especially if iron is scarce or titrated out by other metals.The gram-negative microaerophilic bacterial pathogen Helicobacter pylori colonizes the mucus layer of the human stomach (12,20). Its hostile ecological niche has necessitated the development of regulatory mechanisms which allow the bacterium to survive unfavorable changes in the environment. Adaptation to the conditions in the gastric mucosa includes acquisition mechanisms that overcome a temporary lack of the metals iron and nickel. Iron is essential for maintaining the basic energy and redox metabolism, whereas nickel is an essential cofactor of urease, an important virulence determinant of H. pylori (21). However, as overacquisition of iron, nickel, and other metals is deleterious, the control mechanisms regulating the intracellular availability of these metals are of crucial importance.Iron-responsive regulation in prokaryotes is usually mediated through the ferric uptake regulator (Fur) protein. Fur homologs downregulate the expression of genes involved in iron uptake when the cytoplasmatic ferrous iron concentration increases, thus abolishing iron acquisition (24). Fur homologs using metal ions as cofactors have been identified in many bacterial species (7,9,13,16,19,23,30,32,33), and their regulatory functions range from regulation of metal uptake to more specific processes, like oxidative-stress defense, production of virulence factors, and acid resistance. That the Fur protein also activates transcription in response to iron was recently shown for the iron-induced superoxide dismutase SodB of Escherichia coli (11,13).In addition to regulation of iron uptake, the internal iron concentration within the cell can be modulated by the removal of free ionic iron from the cytoplasm. This function is catalyzed by bacterial ferritins, which constitute a specialized intracellu-

The complete amino acid sequence of cat muscle pyruvate kinase has been determined and fitted to the 2.6 A resolution electron density map. Residues in the active site region are highly conserved in the cat muscle, chicken muscle, rat liver and yeast enzymes. The enzyme‐bound magnesium, which is essential for activity, interacts with the side chain of glutamate‐271 and with two main carbonyl groups. Lysine‐269 is the probable acid/base catalyst responsible for the interconversion of pyruvate and enolpyruvate. A possible binding site for the essential monovalent cation is proposed.

A cell-free system was developed that allows the correct integration of single and multispanning membrane proteins of Escherichia coli into proteoliposomes. We found that physiological levels of diacylglycerol were required to prevent spontaneous integration into liposomes even of the polytopic mannitol permease. Using diacylglycerol-containing proteoliposomes, we identified a novel integration-stimulating factor. Integration of mannitol permease was dependent on both the SecYEG translocon and this factor and was mediated by signal recognition particle and signal recognition particle receptor. Integration of M13 procoat, which is independent of both signal recognition particle/signal recognition particle receptor and SecYEG, was also promoted by this factor. Furthermore, the factor stimulated the post-translational translocation of presecretory proteins, suggesting that it also mediates integration of a signal sequence. This factor was found to be a lipid A-derived membrane component possessing a peptide moiety.

Volume 2g0, number 2, 379~3~ FEELS 09~)4 '~ 1991 Feder#tton of I~aroF~an ltio,~heml¢~l So~iett,¢i 0014~17~1~'91t$] Tile structure of the perth front Rlmd.b~trt¢r ¢cJpxlqhuu~ was determin¢d at a resolution of !,~ A. The analysi=t stalled from a eJt~sety r~laled ¢rytl¢il ~tructur~ thai had been sol~'ed at a medium resolution of 3 A using mulliple isotnorphou~ replacement ttnd solvent llattenin//,. The new structure contains the complete seq uent:e of.~0 ! amino acid residues, Refinement of the model is uoder ~¢~y; the present R.fa¢ior i~ 22,~ with ~ood geometry,Except for the len~lths of several loops, the resultinl~ chain fold corresponds to the medium r0soltttion model, The membrane cha,mel is lined by a larl/e naml:er of ionol|cnic side chains with ¢:haracttriliie segrel~ation ofdifl'~rently charged ~troups.

YvoA is a GntR/HutC transcription regulator from Bacillus subtilis implicated in the regulation of genes from the N-acetylglucosamine-degrading pathway. Its 2.4-Å crystal structure reveals a homodimeric assembly with each monomer displaying a two-domain fold. The C-terminal domain, which binds the effector N-acetylglucosamine-6-phosphate, adopts a chorismate lyase fold, whereas the N-terminal domain contains a winged helix–turn–helix DNA-binding domain. Isothermal titration calorimetry and site-directed mutagenesis revealed that the effector-binding site in YvoA coincides with the active site of related chorismate lyase from Escherichia coli. The characterization of the DNA- and effector-binding properties of two disulfide-bridged mutants that lock YvoA in two distinct conformational states provides for the first time detailed insight into the allosteric mechanism through which effector binding modulates DNA binding and, thereby regulates transcription in a representative GntR/HutC family member. Central to this allosteric coupling mechanism is a loop-to-helix transition with the dipole of the newly formed helix pointing toward the phosphate of the effector. This transition goes in hand with the emergence of internal symmetry in the effector-binding domain and, in addition, leads to a 122° rotation of the DNA-binding domains that is best described as a jumping-jack-like motion.