Flavoenzyme catalysed oxidation of amines: roles for flavin and protein-based radicals.

Amines are a carbon source for the growth of a number of bacterial species and they also play key roles in neurotransmission, cell growth and differentiation, and neoplastic cell proliferation. Enzymes have evolved to catalyse these reactions and these oxidoreductases can be grouped into the flavoprotein and quinoprotein families. The mechanism of amine oxidation catalysed by the quinoprotein amine oxidases is understood reasonably well and occurs through the formation of enzyme-substrate covalent adducts with TPQ (topaquinone), TTQ (tryptophan tryptophylquinone), CTQ (cysteine tryptophylquinone) and LTQ (lysine tyrosyl quinone) redox centres. Oxidation of amines by flavoenzymes is less well understood. The role of protein-based radicals and flavin semiquinone radicals in the oxidation of amines is discussed.

High concordance from independent studies by the Children's Cancer Group (CCG) and Pediatric Oncology Group (POG) associating favorable prognosis with combined trisomies 4, 10, and 17 in children with NCI Standard-Risk B-precursor Acute Lymphoblastic Leukemia: a Children's Oncology Group (COG) initiative.

Chromosome aberrations have a major role in pediatric acute lymphoblastic leukemia (ALL) risk assignment. The Children's Cancer Group (CCG) and the Pediatric Oncology Group (POG) independently assessed the significance of trisomy for chromosomes 4, 10, and 17 in National Cancer Institute (NCI) Standard- and High-Risk ALL. Data from 1582 (CCG) and 3902 (POG) patients were analyzed. Eight-year event-free survivals (EFS) of 91% (CCG) and 89% (POG) (P < 0.001) were achieved in patients assigned to NCI Standard Risk whose leukemic cells had simultaneous trisomies 4, 10, and 17. Both groups showed the degree of favorable prognostic importance increased with the actual number of favorable trisomies. POG analyses also demonstrated hyperdiploidy (> or =53 chromosomes) was less of an independently significant prognostic factor in the absence of these key trisomies. This finding supported conclusions from previous CCG and POG studies that specific trisomies are more important than chromosome number in predicting outcome in pediatric B-precursor ALL. In NCI Higher Risk patients, the number of favorable trisomies was not prognostically significant, but showed the same trend. Moreover, specific trisomies 4, 10, and 17 remain associated with favorable prognosis in Standard-Risk B-precursor ALL, even in the context of very different treatment approaches between the groups.

Differential coupling through Val-344 and Tyr-442 of trimethylamine dehydrogenase in electron transfer reactions with ferricenium ions and electron transferring flavoprotein.

Modeling studies of the trimethylamine dehydrogenase-electron transferring flavoprotein (TMADH-ETF) electron transfer complex have suggested potential roles for Val-344 and Tyr-442, found on the surface of TMADH, in electronic coupling between the 4Fe-4S center of TMADH and the FAD of ETF. The importance of these residues in electron transfer, both to ETF and to the artificial electron acceptor, ferricenium (Fc(+)), has been studied by site-directed mutagenesis and stopped-flow spectroscopy. Reduction of the 6-(S)-cysteinyl FMN in TMADH is not affected by mutation of either Tyr-442 or Val-344 to a variety of alternate side chains, although there are modest changes in the rate of internal electron transfer from the 6-(S)-cysteinyl FMN to the 4Fe-4S center. The kinetics of electron transfer from the 4Fe-4S center to Fc(+) are sensitive to mutations at position 344. The introduction of smaller side chains (Ala-344, Cys-344, and Gly-344) leads to enhanced rates of electron transfer, and likely reflects shortened electron transfer "pathways" from the 4Fe-4S center to Fc(+). The introduction of larger side chains (Ile-344 and Tyr-344) reduces substantially the rate of electron transfer to Fc(+). Electron transfer to ETF is not affected, to any large extent, by mutation of Val-344. In contrast, mutation of Tyr-442 to Phe, Leu, Cys, and Gly leads to major reductions in the rate of electron transfer to ETF, but not to Fc(+). The data indicate that electron transfer to Fc(+) is via the shortest pathway from the 4Fe-4S center of TMADH to the surface of the enzyme. Val-344 is located at the end of this pathway at the bottom of a small groove on the surface of TMADH, and Fc(+) can penetrate this groove to facilitate good electronic coupling with the 4Fe-4S center. With ETF as an electron acceptor, the observed rate of electron transfer is substantially reduced on mutation of Tyr-442, but not Val-344. We conclude that the flavin of ETF does not penetrate fully the groove on the surface of TMADH, and that electron transfer from the 4Fe-4S center to ETF may involve a longer pathway involving Tyr-442. Mutation of Tyr-442 likely disrupts electron transfer by perturbing the interaction geometry of TMADH and ETF in the productive electron transfer complex, leading to less efficient coupling between the redox centers.

Structural and biochemical characterization of recombinant wild type and a C30A mutant of trimethylamine dehydrogenase from methylophilus methylotrophus (sp. W(3)A(1)).

Trimethylamine dehydrogenase (TMADH) is an iron-sulfur flavoprotein that catalyzes the oxidative demethylation of trimethylamine to form dimethylamine and formaldehyde. It contains a unique flavin, in the form of a 6-S-cysteinyl FMN, which is bent by approximately 25 degrees along the N5-N10 axis of the flavin isoalloxazine ring. This unusual conformation is thought to modulate the properties of the flavin to facilitate catalysis, and has been postulated to be the result of covalent linkage to Cys-30 at the flavin C6 atom. We report here the crystal structures of recombinant wild-type and the C30A mutant TMADH enzymes, both determined at 2.2 A resolution. Combined crystallographic and NMR studies reveal the presence of inorganic phosphate in the FMN binding site in the deflavo fraction of both recombinant wild-type and C30A proteins. The presence of tightly bound inorganic phosphate in the recombinant enzymes explains the inability to reconstitute the deflavo forms of the recombinant wild-type and C30A enzymes that are generated in vivo. The active site structure and flavin conformation in C30A TMADH are identical to those in recombinant and native TMADH, thus revealing that, contrary to expectation, the 6-S-cysteinyl FMN link is not responsible for the 25 degrees butterfly bending along the N5-N10 axis of the flavin in TMADH. Computational quantum chemistry studies strongly support the proposed role of the butterfly bend in modulating the redox properties of the flavin. Solution studies reveal major differences in the kinetic behavior of the wild-type and C30A proteins. Computational studies reveal a hitherto, unrecognized, contribution made by the S(gamma) atom of Cys-30 to substrate binding, and a role for Cys-30 in the optimal geometrical alignment of substrate with the 6-S-cysteinyl FMN in the enzyme active site.

The possible role of a disulphide bond in forming functional Kir2.1 potassium channels.

The role of two cysteine residues--Cys122 and Cys154--in the structure of the strong inward rectifier K+ channel, Kir2.1, has been investigated using site-directed mutagenesis and electrophysiology. Such cysteine residues are conserved across the inward rectifier family and may be expected to form a crucial disulphide bond. Our experiments show that when the cysteines are absent, the protein is expressed, but the channels are not functional, suggesting that the disulphide bond is essential for correct channel assembly. However, reducing agents applied extracellularly have little effect on current amplitude in wild-type, so that, once the channel is assembled correctly in the membrane, the disulphide bonds are no longer essential for function. Molecular modelling suggests that a disulphide bond is formed--this may be either an intra- or an inter-subunit.

Cysteine mutagenesis and homology modeling of the ligand-binding site of a kainate-binding protein.

Glutamate receptors comprise the most abundant group of neurotransmitter receptors in the vertebrate central nervous system. Cysteine mutagenesis in combination with homology modeling has been used to study the determinants of kainate binding in a glutamate receptor subtype, a low molecular weight goldfish kainate-binding protein, GFKARbeta. A construct of GFKARbeta with no cysteines in the extracellular domain was produced, and single cysteine residues were introduced at selected positions. N-Ethylmaleimide or derivatized methanethiosulfonate reagents (neutral or charged) were used to modify the introduced cysteines covalently, and the effect on [(3)H]kainate binding was determined. In addition, cysteine mutants of GFKARbeta transiently expressed in HEK293 cells were labeled with a membrane-impermeable biotinylating reagent followed by precipitation with streptavidin beads and specific detection of GFKARbeta by Western blot analysis. The results are consistent with the proposal that the energy driving kainate binding is contributed both from residues within the binding site and from interactions between two regions (i.e. two lobes) of the protein that are brought into contact upon ligand binding in a manner analogous to that seen in bacterial amino acid-binding proteins.

Identification of the binding surface on Cdc42Hs for p21-activated kinase.

The Ras superfamily of GTP-binding proteins is involved in a number of cellular signaling events including, but not limited to, tumorigenesis, intracellular trafficking, and cytoskeletal organization. The Rho subfamily, of which Cdc42Hs is a member, is involved in cell morphogenesis through a GTPase cascade which regulates cytoskeletal changes. Cdc42Hs has been shown to stimulate DNA synthesis as well as to initiate a protein kinase cascade that begins with the activation of the p21-activated serine/threonine kinases (PAKs). We have determined previously the solution structure of Cdc42Hs [Feltham et al. (1997) Biochemistry 36, 8755-8766] using NMR spectroscopy. A minimal-binding domain of 46 amino acids of PAK was identified (PBD46), which binds Cdc42Hs with a KD of approximately 20 nM and inhibits GTP hydrolysis. The binding interface was mapped by producing a fully deuterated sample of 15N-Cdc42Hs bound to PBD46. A 1H,15N-NOESY-HSQC spectrum demonstrated that the binding surface on Cdc42Hs consists of the second beta-strand (beta2) and a portion of the loop between the first alpha-helix (alpha1) and beta2 (switch I). A complex of PBD46 bound to 15N-Cdc42Hs.GMPPCP exhibited extensive chemical shift changes in the 1H,15N-HSQC spectrum. Thus, PBD46 likely produces structural changes in Cdc42Hs which are not limited to the binding interface, consistent with its effects on GTP hydrolysis. These results suggest that the kinase-binding domain on Cdc42Hs is similar to, but more extensive than, the c-Raf-binding domain on the Ras antagonist, Rap1 [Nassar et al. (1995) Nature 375, 554-560)].

Pre-B acute lymphoblastic leukemia in a 3-year-old boy with pre-acute myelogenous leukemia myelodysplastic syndrome: cytogenetic evidence of common early progenitor cell ontogeny.

PURPOSE: Myelodysplastic syndromes in children commonly evolve into acute leukemia, usually acute myelogenous leukemia (AML) and rarely acute lymphoblastic leukemia (ALL). The lineage of the leukemia can be predicted based on characteristic morphologic and cytogenetic findings of the marrow and peripheral blood. PATIENT AND METHODS: A 3-year-old boy had refractory anemia with excess blasts and abnormalities suggestive of pre-AML with highly unusual cytogenetic changes. ALL of pre-B phenotype developed. RESULTS: Leukoerythroblastic anemia, pseudo Pelger-Huet neutrophils, and dysmyelopoietic hyperplasia of the marrow suggested likely early progression to AML. Complex cytogenetic abnormalities (monosomy 17 and 20, ring chromosome 11 with deletion of bands q23, and a derivative dicentric chromosome 12) were present in both the myelodysplastic marrow and the subsequent ALL. CONCLUSION: This case presents cytogenetic evidence of common early progenitor cell ontogeny of both malignancies (refractory anemia with excess blasts and ALL).

The dependence of Ag+ block of a potassium channel, murine kir2.1, on a cysteine residue in the selectivity filter.

Externally applied Ag+ (100-200 nM) irreversibly blocked the strong inwardly rectifying K+ channel, Kir2.1. Mutation to serine of a cysteine residue at position 149 in the pore-forming H5 region of Kir2.1 abolished Ag+ blockage. To determine how many of the binding sites must be occupied by Ag+ before the channel is blocked, we measured the rate of channel block and found that our results were best fitted assuming that only one Ag+ ion need bind to eliminate channel current. We tested our hypothesis further by constructing covalently linked dimers and tetramers of Kir2.1 in which cysteine had been replaced by serine in one (dimer) or three (tetramer) of the linked subunits. When expressed, these constructs yielded functional channels with either two (dimer) or one (tetramer) cysteines per channel at position 149. Blockage in the tetramer was complete after sufficient exposure to 200 nM Ag+, a result that is also consistent with only one Ag+ being required to bind to Cys149 to block fully. The rate of development of blockage was 16 times slower than in wild-type channels; the rate was 4 times slower in channels formed from dimers.

The selectivity filter of a potassium channel, murine kir2.1, investigated using scanning cysteine mutagenesis.

We have produced a structural model of the pore-forming H5 (or P) region of the strong inward rectifier K+ channel, Kir2.1, based initially on an existing molecular model of the pore region of the voltage-gated K+ channel, Kv1.3. Cysteine-scanning mutagenesis and subsequent blockage by Ag+ was used to test our model by determining the residues in H5 whose side chains line the ion conduction pathway. Mutations made in eight positions within the highly conserved H5 region resulted in apparently non-functional channels. Constructing covalently linked dimers, which carry a cysteine substitution in only one of the linked subunits, rescued six of these mutants; a covalently linked tetramer, carrying a cysteine substitution on only one of the linked subunits, rescued a further mutant. Our results using the dimers and tetramers suggest that residues Thr141, Thr142, Ile143, Tyr145, Phe147 and Cys149 are accessible to externally applied Ag+ (100-200 nM) and therefore that their side chains line the channel pore. We conclude that the topology of the Kir pore is similar, but not identical, to that of Kv channels. Additionally, the molecular model suggests that selectivity may be conferred both by aromatic residues (Tyr145 and Phe147) via cation-pi interactions and by backbone carbonyl groups (Thr142 and Gly144).

Determinants of the substrate specificity of human cytochrome P-450 CYP2D6: design and construction of a mutant with testosterone hydroxylase activity.

Cytochrome P-450 CYP2D6, human debrisoquine hydroxylase, metabolizes more than 30 prescribed drugs, the vast majority of which are small molecules containing a basic nitrogen atom. In contrast, the similar mouse protein Cyp2d-9 was first characterized as a testosterone 16alpha-hydroxylase. No common substrates have been reported for the two enzymes. Here we investigate the structural basis of this difference in substrate specificity. We have earlier used a combination of NMR data and homology modelling to generate a three-dimensional model of CYP2D6 [Modi, Paine, Sutcliffe, Lian, Primrose, Wolf, C.R. and Roberts (1996) Biochemistry 35, 4541-4550]. We have now generated a homology model of Cyp2d-9 and compared the two models to identify specific amino acid residues that we believe form the substrate-binding site in each protein and therefore influence catalytic selectivity. Although there are many similarities in active site structure, the most notable difference is a phenylalanine residue (Phe-483) in CYP2D6, which in the model is located such that the bulky phenyl ring is positioned across the channel mouth, thus limiting the size of substrate that can access the active site. In Cyp2d-9, the corresponding position is occupied by an isoleucine residue, which imposes fewer steric restraints on the size of substrate that can access the active site. To investigate whether the amino acid residue at this position does indeed influence the catalytic selectivity of these enzymes, site-directed mutagenesis was used to change Phe-483 in CYP2D6 to isoleucine and also to tryptophan. CYP2D6, Cyp2d-9 and both mutant CYP2D6 proteins were co-expressed with NADPH cytochrome P-450 reductase as a functional mono-oxygenase system in Escherichia coli and their relative catalytic activities towards bufuralol and testosterone were determined. All four proteins exhibited catalytic activity towards bufuralol but only Cyp2d-9 catalysed the formation of 16alpha-hydroxytesterone. Uniquely, the CYP2D6F483I mutant acquired the ability to metabolize testosterone to a novel product, which was identified by MS and proton NMR spectroscopy as 15alpha-hydroxytestosterone. NMR spin relaxation experiments were used to measure distances between the haem iron and protons of testosterone bound to the CYP2D6F483I mutant. These experiments demonstrate that very minor modifications to the active site structure of CYP2D6 can have a profound influence on the substrate specificity of the enzyme.

Crystal structure of a PDZ domain.

PDZ domains (also known as DHR domains or GLGF repeats) are approximately 90-residue repeats found in a number of proteins implicated in ion-channel and receptor clustering, and the linking of receptors to effector enzymes. PDZ domains are protein-recognition modules; some recognize proteins containing the consensus carboxy-terminal tripeptide motif S/TXV with high specificity. Other PDZ domains form homotypic dimers: the PDZ domain of the neuronal enzyme nitric oxide synthase binds to the PDZ domain of PSD-95, an interaction that has been implicated in its synaptic association. Here we report the crystal structure of the third PDZ domain of the human homologue of the Drosophila discs-large tumour-suppressor gene product, DlgA. It consists of a five-stranded antiparallel beta-barrel flanked by three alpha-helices. A groove runs over the surface of the domain, ending in a conserved hydrophobic pocket and a buried arginine; we suggest that this is the binding site for the C-terminal peptide.

Three-dimensional structure of the RGD-containing neurotoxin homologue dendroaspin.

Dendroaspin is a short chain neurotoxin homologue from the venom of Elapidae snakes, which lacks neurotoxicity. Unlike neurotoxins, it contains an Arg-Gly-Asp-(RGD)-motif and functions as an inhibitor of platelet aggregation and platelet adhesion with comparable potency to the disintegrins from the venoms of Viperidae. We have determined the structure of dendroaspin in solution using NMR spectroscopy. The structure contains a core similar to that of short chain neurotoxins, but with a novel arrangement of loops and a solvent-exposed RGD-motif. Dendroaspin is thus an integrin antagonist with a well defined fold different from that of the disintegrins, based on the neurotoxin scaffold.

A single aspartate residue is involved in both intrinsic gating and blockage by Mg2+ of the inward rectifier, IRK1.

1. We describe the effects on channel function of changing an aspartate residue (Asp172) in a membrane-spanning alpha-helix of the murine inward rectifier, IRK1, by site-directed mutagenesis. 2. Alteration of Asp172 to Glu (charged) or to Gln or Asn (polar but uncharged) produced functional channels showing inward rectification, though rectification was weaker with Gln and Asn. 3. Intrinsic gating around the potassium equilibrium potential, EK, was conserved only if the charge on residue 172 was conserved. Currents through channels with Gln or Asn in this position showed no time dependence under hyperpolarization. 4. The change from Asp to Gln also reduced the affinity for internal Mg2+ at least fivefold, indicating that Asp172 also forms part of the site for Mg2+ blockage. 5. The consequences for channel structure of Asp172 lining the pore are discussed.

Sequence-specific 1H NMR assignments and solution structure of bovine pancreatic polypeptide.

Sequence-specific 1H NMR assignments for the 36 residue bovine pancreatic polypeptide (bPP) have been completed. The secondary and tertiary structure of bPP in solution has been determined from experimental NMR data. It is shown that bPP has a very well-defined C-terminal alpha-helix involving residues 15-32. Although regular secondary structure cannot be clearly defined in the N-terminal region, residues 4-8 maintain a rather ordered conformation in solution. This is attributed primarily to the hydrophobic interactions between this region and the C-terminal helix. The two segments of the structure are joined by a turn which is poorly defined. The four end residues both at the N-terminus and the C-terminus are highly disordered in solution. The overall fold of the bPP molecule is very closely similar to that found in the crystal structure of avian pancreatic polypeptide (aPP). The RMS deviation for backbone atoms of residues 4-8 and 15-32 between the bPP mean structure and the aPP crystal structure is 0.65 A, although there is only 39% identity of the residues. Furthermore, the average conformations of some (mostly from the alpha-helix) side chains of bPP in solution are closely similar to those of aPP in the crystal structure. A large number of side chains of bPP, however, show significant conformational averaging in solution.