Two novel germline KRAS mutations: expanding the molecular and clinical phenotype.

Noonan and Cardio-facio-cutaneous (CFC) syndromes are characterized by typical dysmorphic features, cardiac defects, short stature, variable ectodermal anomalies, and intellectual disability. Both belong to the Ras/mitogen-activated protein kinase pathway group of disorders and clinical features overlap other related conditions, notably LEOPARD and Costello syndromes. KRAS mutations account for about 2% of reported Noonan and <5% of reported CFC cases. The mutation spectrum includes recurrent missense changes clustering in particular domains of the KRAS protein and conferring gain-of-function. We report three patients from two unrelated families with novel missense KRAS mutations, p.K147E and p.Y71H. Both mutations affect a residue which is highly conserved in KRAS and other RAS isoforms. One of the families includes a mother and son pair who represent the first report of a vertically transmitted KRAS mutation. In addition, the mother and son pair had peripheral neuropathy, complicated by Charcot arthropathy in the mother. An unusual phenotypic effect of the specific KRAS mutation or a coincidence of two independent disorders may be considered. KRAS mutation-associated phenotypes appear to be subject to considerable clinical heterogeneity. All three cases highlight the challenges of clinical assessment in KRAS mutation-positive patients, and the utility of molecular testing as an adjunct to diagnosis.

Crystal structure of a carbon monoxide dehydrogenase reveals a [Ni-4Fe-5S] cluster.

The homodimeric nickel-containing CO dehydrogenase from the anaerobic bacterium Carboxydothermus hydrogenoformans catalyzes the oxidation of CO to CO2. A crystal structure of the reduced enzyme has been solved at 1.6 angstrom resolution. This structure represents the prototype for Ni-containing CO dehydrogenases from anaerobic bacteria and archaea. It contains five metal clusters of which clusters B, B', and a subunit-bridging, surface-exposed cluster D are cubane-type [4Fe-4S] clusters. The active-site clusters C and C' are novel, asymmetric [Ni-4Fe-5S] clusters. Their integral Ni ion, which is the likely site of CO oxidation, is coordinated by four sulfur ligands with square planar geometry.

The role of Se, Mo and Fe in the structure and function of carbon monoxide dehydrogenase.

CO dehydrogenase (EC 1.2.99.2) catalyzes the oxidation of CO according to the following equation: CO + H2O-->CO2 + 2 e- + 2 H+. It is a selenium-containing molybdo-iron-sulfur-flavoenzyme, which has been crystallized and structurally characterized in its oxidized state from the aerobic CO utilizing bacteria Oligotropha carboxidovorans and Hydrogenophaga pseudoflava. Both CO dehydrogenase structures show only minor differences, and the enzymes are dimers of two heterotrimers. Each heterotrimer is composed of a molybdoprotein, a flavoprotein, and an iron-sulfur protein. CO oxidation takes place at the molybdoprotein which contains a 1:1 mononuclear complex of molybdopterin-cytosine dinucleotide and a Mo-ion, along with a catalytically essential S-selanylcysteine. The latter is appropriately positioned in the SeMo-active site by a unique VAYRCSFR active site loop. In H. pseudoflava the arginine preceeding the cysteine in the active site loop is modified to a Cgamma-hydroxy arginine residue which has no obvious function. The substituents in the first coordination sphere of the Mo-ion are the enedithiolate sulfur atoms of the molybdopterin-cytosine dinucleotide, two oxo- and a sulfido-group. Extended X-ray absorption fine structure spectroscopy (EXAFS), along with the crystal structure of CO dehydrogenase (23.2 U mg(-1)) at 1.85 A resolution, have identified a sulfur atom at 2.3 A from the Mo-ion. The sulfur reacts with cyanide yielding thiocyanate. The corresponding inactive desulfo-CO dehydrogenase shows a typical desulfo inhibited-type of Mo-electron paramagnetic resonance (EPR) spectrum. Structural changes at the SeMo-site during catalysis are suggested by the Mo to Se distance of 3.7 A and the Mo-S-Se angle of 113 degrees in the oxidized enzyme which increase to 4.1 A, and 121 degrees, respectively, in the reduced enzyme. The intramolecular electron transport chain in CO dehydrogenase involves the following prosthetic groups and minimal distances: CO-->[Mo of the molybdenum cofactor] - 14.6 A - [2Fe-2S]I - 12.4 A - [2Fe-2S]II - 8.7 A - [FAD].

The effect of intracellular molybdenum in Hydrogenophaga pseudoflava on the crystallographic structure of the seleno-molybdo-iron-sulfur flavoenzyme carbon monoxide dehydrogenase.

Crystal structures of carbon monoxide dehydrogenase (CODH), a seleno-molybdo-iron-sulfur flavoprotein from the aerobic carbon monoxide utilizing carboxidotrophic eubacterium Hydrogenophaga pseudoflava, have been determined from the enzyme synthesized at high (Mo(plus) CODH) and low intracellular molybdenum content (Mo(minus) CODH) at 2.25 A and 2.35 A resolution, respectively. The structures were solved by Patterson search methods utilizing the enzyme from Oligotropha carboxidovorans as the initial model. The CODHs from both sources are structurally very much conserved and show the same overall fold, architecture and arrangements of the molybdopterin-cytosine dinucleotide-type of molybdenum cofactor, the type I and type II [2Fe-2S] clusters and the flavin-adenine dinucleotide. Unlike the CODH from O. carboxidovorans, the enzyme from H. pseudoflava reveals a unique post-translationally modified C(gamma)-hydroxy-Arg384 residue which precedes the catalytically essential S-selanyl-Cys385 in the active-site loop. In addition, the Trp193 which shields the isoalloxazine ring of the flavin-adenine dinucleotide in the M subunit of the H. pseudoflava CODH is a Tyr193 in the O. carboxidovorans CODH. The hydrogen bonding interaction pattern of the molybdenum cofactor involves 27 hydrogen bonds with the surrounding protein. Of these, eight are with the cytosine moiety, eight with the pyrophosphate, six with the pyranopterin, and five with the ligands of the Mo ion. The structure of the catalytically inactive Mo(minus) CODH indicates that an intracellular Mo-deficiency affects exclusively the active site of the enzyme as an incomplete non-functional molybdenum cofactor was synthesized. The 5'-CDP residue was present in Mo(minus) CODH, whereas the Mo-pyranopterin moiety was absent. In Mo(plus) CODH the selenium faces the Mo ion and flips away from the Mo site in Mo(minus) CODH. The different side-chain conformations of the active-site residues S-selanyl-Cys385 and Glu757 in Mo(plus) and Mo(minus) CODH indicate a side-chain flexibility and a function of the Mo ion in the proper orientation of both residues.

Binding of flavin adenine dinucleotide to molybdenum-containing carbon monoxide dehydrogenase from Oligotropha carboxidovorans. Structural and functional analysis of a carbon monoxide dehydrogenase species in which the native flavoprotein has been replaced by its recombinant counterpart produced in Escherichia coli.

The carbon monoxide (CO) dehydrogenase of Oligotropha carboxidovorans is composed of an S-selanylcysteine-containing 88. 7-kDa molybdoprotein (L), a 17.8-kDa iron-sulfur protein (S), and a 30.2-kDa flavoprotein (M) in a (LMS)(2) subunit structure. The flavoprotein could be removed from CO dehydrogenase by dissociation with sodium dodecylsulfate. The resulting M(LS)(2)- or (LS)(2)-structured CO dehydrogenase species could be reconstituted with the recombinant apoflavoprotein produced in Escherichia coli. The formation of the heterotrimeric complex composed of the apoflavoprotein, the molybdoprotein, and the iron-sulfur protein involves structural changes that translate into the conversion of the apoflavoprotein from non-FAD binding to FAD binding. Binding of FAD to the reconstituted deflavo (LMS)(2) species occurred with second-order kinetics (k(+1) = 1350 M(-1) s(-1)) and high affinity (K(d) = 1.0 x 10(-9) M). The structure of the resulting flavo (LMS)(2) species at a 2.8-A resolution established the same fold and binding of the flavoprotein as in wild-type CO dehydrogenase, whereas the S-selanylcysteine 388 in the active-site loop on the molybdoprotein was disordered. In addition, the structural changes related to heterotrimeric complex formation or FAD binding were transmitted to the iron-sulfur protein and could be monitored by EPR. The type II 2Fe:2S center was identified in the N-terminal domain and the type I center in the C-terminal domain of the iron-sulfur protein.

Crystal structure and mechanism of CO dehydrogenase, a molybdo iron-sulfur flavoprotein containing S-selanylcysteine.

CO dehydrogenase from the aerobic bacterium Oligotropha carboxidovorans catalyzes the oxidation of CO with H(2)O, yielding CO(2), two electrons, and two H(+). Its crystal structure in the air-oxidized form has been determined to 2.2 A. The active site of the enzyme, which contains molybdenum with three oxygen ligands, molybdopterin-cytosine dinucleotide and S-selanylcysteine, delivers the electrons to an intramolecular electron transport chain composed of two types of [2Fe-2S] clusters and flavin-adenine dinucleotide. CO dehydrogenase is composed of an 88.7-kDa molybdoprotein (L), a 30. 2-kDa flavoprotein (M), and a 17.8-kDa iron-sulfur protein (S). It is organized as a dimer of LMS heterotrimers and resembles xanthine dehydrogenase/oxidase in many, but not all, aspects. A mechanism based on a structure with the bound suicide-substrate cyanide is suggested and displays the necessity of S-selanylcysteine for the catalyzed reaction.

Characterization of xanthine dehydrogenase from the anaerobic bacterium Veillonella atypica and identification of a molybdopterin-cytosine-dinucleotide-containing molybdenum cofactor.

The molybdenum-containing iron-sulfur flavoprotein xanthine dehydrogenase from the anaerobic bacterium Veillonella atypica has been purified approximately 800-fold with a yield of approximately 40% and a specific activity of approximately 70 micromol ferricyanide reduced x min(-1) x mg protein(-1) with xanthine as electron donor, which corresponds to approximately 30 micromol xanthine oxidized x min(-1) x mg protein(-1) with methylene blue as electron acceptor. The 129-kDa enzyme was a non-covalent heterotrimer with large (82.4 kDa), medium (28.5 kDa) and small (18.4 kDa) subunits. The N-termini of the small and medium polypeptides of V. atypica xanthine dehydrogenase and the corresponding domains of eukaryotic xanthine dehydrogenases were similar, whereas the N-terminus of the large polypeptide was unrelated to eukaryotic xanthine dehydrogenases. The enzyme contained 0.86 atoms Mo, 1.75 atoms Fe, 1.61 atoms acid-labile sulfur and 0.68 molecules FAD/molecule, which corresponds to a 1:2.0:1.9:0.8 molar ratio. Acid hydrolysis revealed 0.95 mol CMP and 0.80 mol AMP/mol xanthine dehydrogenase. After treatment of the enzyme with iodoacetamide, di(carboxamidomethyl)molybdopterin cytosine dinucleotide was identified, which indicates that molybdopterin cytosine dinucleotide is the organic portion of the V. atypica xanthine dehydrogenase molybdenum cofactor. The enzyme and its molybdenum cofactor occurred in a 1:1 molar ratio. Xanthine dehydrogenases from eukaryotic sources are characterized by a domain structure and the presence of duplicate copies of two types of [2Fe-2S) clusters. In contrast, the xanthine dehydrogenase from V. atypica had a heterotrimeric subunit structure and a single [2Fe-2S] cluster. In addition, the enzyme indicates the presence of a molybdopterin dinucleotide as a constituent of a xanthine dehydrogenase molybdenum cofactor.