Crystal structure of Methanobacterium thermoautotrophicum phosphoribosyl-AMP cyclohydrolase HisI

The metabolic pathway for histidine biosynthesis is interesting from an evolutionary perspective because of the diversity of gene organizations and protein structures involved. Hydrolysis of phosphoribosyl-AMP, the third step in the histidine biosynthetic pathway, is carried out by PR-AMP cyclohydrolase, the product of the hisI gene. The three-dimensional structure of PR-AMP cyclohydrolase from Methanobacterium thermoautotrophicum was solved and refined to 1.7 A resolution. The enzyme is a homodimer. The position of the Zn(2+)-binding site that is essential for catalysis was inferred from the positions of bound Cd(2+) ions, which were part of the crystallization medium. These metal binding sites include three cysteine ligands, two from one monomer and the third from the second monomer. The enzyme remains active when Cd(2+) is substituted for Zn(2+). The likely binding site for Mg(2+), also necessary for activity in a homologous cyclohydrolase, was also inferred from Cd(2+) positions and is comprised of aspartic acid side chains. The putative substrate-binding cleft is formed at the interface between the two monomers of the dimer. This fact, combined with the localization of the Zn(2+)-binding site, indicates that the enzyme is an obligate dimer.     

Redox signaling in central neural regulation of cardiovascular function

One of the most prominent concepts to emerge in cardiovascular research over the past decade, especially in areas focused on angiotensin II (AngII), is that reactive oxygen species (ROS) are critical signaling molecules in a wide range of cellular processes. Many of the physiological effects of AngII are mediated by ROS, and alterations in AngII-mediated redox mechanisms are implicated in cardiovascular diseases such as hypertension and atherosclerosis. Although most investigations to date have focused on the vasculature as a key player, the nervous system has recently begun to gain attention in this field. Accumulating evidence suggests that ROS have important effects on central neural mechanisms involved in blood pressure regulation, volume homeostasis, and autonomic function, particularly those that involve AngII signaling. Furthermore, oxidant stress in the central nervous system is implicated in the neuro-dysregulation associated with some forms of hypertension and heart failure. The main objective of this review is to discuss the recent progress and prospects for this new field of central redox signaling in cardiovascular regulation, while also addressing the molecular tools that have spurred it forward.     

Scrambler,a new neurological mutation of the mouse with abnormalities of neuronal migration

A novel spontaneous neurological mutation, scrambler (scm), appeared in the inbred mouse strain DC/Le (dancer) in 1991. Mice homozygous for this recessive mutation are recognized by an unstable gait and whole-body tremor. The cerebella of 30-day-old scrambler homozygotes are hypoplastic and devoid of folia; however, neither seizures nor abnormal brain wave patterns have been observed. Homozygous scrambler mutants have an ataxic gait which in the male may be a contributory factor in the failure to mate. Female homozygotes mate and breed. Life span is not reduced in either sex. Scrambler is similar to the reeler mutation in phenotype and pathology and, like reeler, probably results from defective neuronal migration. We mapped the scrambler mutation to Chromosome (Chr) 4, proving that it is distinct from the recently cloned reeler gene on Chr 5. We also determined the map position of the agrin gene, Agrn, on Chr 4, and on this basis eliminated it as a candidate for scm. Currently there is no known homology of scrambler with human lissencephalies or other human disorders caused by abnormal neuronal migration. Received: 13 May 1996 / Accepted: 10 July 1996     

Effect of the mouse scid mutation on meiotic recombination

The goal of this study was to determine the effect of the mouse severe combined immunodeficiency (scid) mutation on the rate of meiotic recombination, by standard backcross linkage analysis. For this purpose, we examined four crosses that involved F₁ hybrid animals heterozygous for the strain C57BL/6 and BALB/c genomes. In one set of reciprocal crosses, F₁ animals were homozygous scid/scid, and in a second set of reciprocal crosses, F₁ mice were homozygous wild-type (+/+) at the scid locus. Backcross progeny were typed for recombination between selected genetic markers on mouse Chromosomes (Chrs) 1, 4, 6, 7, 9, 15, and 17. Although some differences in recombination were observed over some intervals, the expression of the SCID phenotype did not appear to have a major or consistent effect on meiotic recombination. Received: 4 October 1995 / Accepted: 2 April 1996     

Preliminary x-ray analysis of Escherichia coli GMP synthetase: Determination of anomalous scattering factors for a cysteinyl mercury derivative

We have initiated a project to determine the three-dimensional structure of GMP synthetase (GMPS) from Escherichia coli. GMPS catalyzes the conversion of XMP to GMP in the final step of de novo guanine nucleotide biosynthesis, and is a member of the glutamine amidotransferase family: a group of enzymes responsible for the assimilation of nitrogen into compounds such as amino acids, purine and pyrimidine bases, amino sugars, and antibiotics. The E. coli guaA gene encoding GMPS was cloned into a tac expression vector, overexpressed, and its gene product purified. Conditions for the growth of protein crystals were developed using recombinant GMPS in the presence of MgCl₂, ATP, and XMP. The crystals are monoclinic, space group P2₁, with cell parameters of a = 156.0 Å, b = 102.0 Å, c = 78.8 Å, β= 96.7°. Diffraction data to 2.8 Å spacings were collected on a Xuong-Hamlin area detector with an overall R_sym of 5.2%. Both the volume of the unit cell and the peaks in the self-rotation function are consistent with one GMPS tetramer of D₂ symmetry in the crystallographic asymmetric unit. Previously, GMPS has been observed only as a dimer in solution. GMPS was covalently modified with p-chloromercuribenzylsulfonic acid (PCMBS), and its X-ray fluorescence spectrum was measured through the L_III absorption edge of mercury Anomalous scattering factors for cysteinyl mercury were derived from this spectrum, and the feasibility of structure determination by multi-wavelength anomalous diffraction was evaluated. The optimal MAD dispersive signal is 4.5% of |F|, and the optimal MAD Bijvoet signal is 7.5% of |F| at a concentration of approximately 1 mercury per 10-kDa protein. The anomalous scattering factors tabulated here should be transferable to cysteinyl mercury in other proteins. © 1994 John Wiley & Sons, Inc.     

Mouse Chromosome 18

Chair of Committee for Mouse Chromosome 18     

Know thy mouse

In science it is imperative that the basic reagents and materials are defined and uniform. Unlike chemical reagents, which are uniform over time, mice, like all living creatures, have an intrinsic genetic drive to change, with mutations accumulating over time leading to increasing genetic variation and phenotypic change. Such changes compromise the reproducibility of experimental data over time and place. The use of the mouse has expanded rapidly in recent years and many scientists who have turned to the mouse as a research model might be unaware of the profound impact of changes in genetic background. Here we discuss the sources of genetic change and strategies to reduce them with the idea of strengthening international genetic standards for inbred mice.     

Catalytic zinc site and mechanism of the metalloenzyme PR-AMP cyclohydrolase

The enzyme N(1)-(5'-phosphoribosyl) adenosine-5'-monophosphate cyclohydrolase (PR-AMP cyclohydrolase) is a Zn(2+) metalloprotein encoded by the hisI gene. It catalyzes the third step of histidine biosynthesis, an uncommon ring-opening of a purine heterocycle for use in primary metabolism. A three-dimensional structure of the enzyme from Methanobacterium thermoautotrophicum has revealed that three conserved cysteine residues occur at the dimer interface and likely form the catalytic site. To investigate the functions of these cysteines in the enzyme from Methanococcus vannielii, a series of biochemical studies were pursued to test the basic hypothesis regarding their roles in catalysis. Inactivation of the enzyme activity by methyl methane thiosulfonate (MMTS) or 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) also compromised the Zn(2+) binding properties of the protein inducing loss of up to 90% of the metal. Overall reaction stoichiometry and the potassium cyanide (KCN) induced cleavage of the protein suggested that all three cysteines were modified in the process. The enzyme was protected from DTNB-induced inactivation by inclusion of the substrate N(1)-(5'-phosphoribosyl)adenosine 5'-monophosphate; (PR-AMP), while Mg(2+), a metal required for catalytic activity, enhanced the rate of inactivation. Site-directed mutations of the conserved C93, C109, C116 and the double mutant C109/C116 were prepared and analyzed for catalytic activity, Zn(2+) content, and reactivity with DTNB. Substitution of alanine for each of the conserved cysteines showed no measurable catalytic activity, and only the C116A was still capable of binding Zn(2+). Reactions of DTNB with the C109A/C116A double mutant showed that C93 is completely modified within 0.5 s. A model consistent with these data involves a DTNB-induced mixed disulfide linkage between C93 and C109 or C116, followed by ejection of the active site Zn(2+) and provides further evidence that the Zn(2+) coordination site involves the three conserved cysteine residues. The C93 reactivity is modulated by the presence of the Zn(2+) and Mg(2+) and substantiates the role of this residue as a metal ligand. In addition, Mg(2+) ligand binding site(s) indicated by the structural analysis were probed by site-directed mutagenesis of three key aspartate residues flanking the conserved C93 which were shown to have a functional impact on catalysis, cysteine activation, and metal (zinc) binding capacity. The unique amino acid sequence, the dynamic properties of the cysteine ligands involved in Zn(2+) coordination, and the requirement for a second metal (Mg(2+)) are discussed in the context of their roles in catalysis. The results are consistent with a Zn(2+)-mediated activation of H(2)O mechanism involving histidine as a general base that has features similar to but distinct from those of previously characterized purine and pyrimidine deaminases.     

Iejimalides A and B inhibit lysosomal vacuolar H+‐ATPase (V‐ATPase) activity and induce S‐phase arrest and apoptosis in MCF‐7 cells

Iejimalides are novel macrolides that are cytostatic or cytotoxic against a wide range of cancer cells at low nanomolar concentrations. A recent study by our laboratory characterized the expression of genes and proteins that determine the downstream effects of iejimalide B. However, little is known about the cellular target(s) of iejimalide or downstream signaling that lead to cell‐cycle arrest and/or apoptosis. Iejimalides have been shown to inhibit the activity of vacuolar H⁺‐ATPase (V‐ATPase) in osteoclasts, but how this inhibition may lead to cell‐cycle arrest and/or apoptosis in epithelial cells is not known. In this study, MCF‐7 breast cancer cells were treated with iejimalide A or B and analyzed for changes in cell‐cycle dynamics, apoptosis, lysosomal pH, cytoplasmic pH, mitochondrial membrane potential, and generation of reactive oxygen species. Both iejimalides A and B sequentially neutralize the pH of lysosomes, induce S‐phase cell‐cycle arrest, and trigger apoptosis in MCF‐7 cells. Apoptosis occurs through a mechanism that involves oxidative stress and mitochondrial depolarization but not cytoplasmic acidification. These data confirm that iejimalides inhibit V‐ATPase activity in the context of epithelial tumor cells, and that this inhibition may lead to a lysosome‐initiated cell death process. J. Cell. Biochem. 109: 634–642, 2010. © 2009 Wiley‐Liss, Inc.