The structure of a molybdopterin precursor. Characterization of a stable, oxidized derivative

An oxidized pterin species, termed compound Z, has been isolated from molybdenum cofactor-deficient mutants of Escherichia coli and shown to be the direct product of oxidation of a molybdopterin precursor which accumulates in these mutants. The complete structural characterization of compound Z has been accomplished. A carbonyl function at C-1' of the 6-alkyl side chain can be reacted with 2,4-dinitrophenylhydrazine to yield a phenylhydrazone and can be reduced with borohydride, producing a mixture of two enantiomers, each with a hydroxyl group on C-1'. Compound Z contains one phosphate/pterin and no sulfur. The phosphate group is insensitive to alkaline phosphatase and to a number of phosphodiesterases but is quantitatively released as inorganic phosphate by mild acid hydrolysis. From 31P and 1H NMR of compound Z it was inferred that the phosphate is bound to C-2' and C-4' of a 4-carbon side chain, forming a 6-membered cyclic structure. Mass spectral analysis showed an MH+ ion with an exact mass of 344.0401 corresponding to the molecular formula C10H11N5O7P, confirming the proposed structure.     

Role of enzyme-bound 5,10-methenyltetrahydropteroylpolyglutamate in catalysis by Escherichia coli DNA photolyase

DNA photolyase catalyzes the photoreversal of pyrimidine dimers. The enzymes from Escherichia coli and yeast contain a flavin chromophore and a folate cofactor, 5,10-methenyltetrahydropteroylpolyglutamate. E. coli DNA photolyase contains about 0.3 mol of folate/mol flavin, whereas the yeast photolyase contains the full complement of folate. E. coli DNA photolyase is reconstituted to a full complement of the folate by addition of 5,10-methenyltetrahydrofolate to cell lysates or purified enzyme samples. The reconstituted enzyme displays a higher photolytic cross section under limiting light. Treatment of photolyase with sodium borohydride or repeated camera flashing results in the disappearance of the absorption band at 384 nm and is correlated with the formation of modified products from the enzyme-bound 5,10-methenyltetrahydrofolate. Photolyase modified in this manner has a decreased photolytic cross section under limiting light. Borohydride reduction results in the formation of 5,10-methylenetetrahydrofolate and 5-methyltetrahydrofolate, both of which are released from the enzyme. Repeated camera flashing results in photodecomposition of the enzyme-bound 5,10-methenyltetrahydrofolate and release of the decomposition products. Finally, it is observed that photolyase binds 10-formyltetrahydrofolate and appears to cyclize it to form the 5,10-methenyltetrahydrofolate chromophore.     

Characterization of Escherichia coli MoeB and its involvement in the activation of molybdopterin synthase for the biosynthesis of the molybdenum cofactor.

Amino acid sequence comparisons of Escherichia coli MoeB suggested that the MoeB-dependent formation of a C-terminal thiocarboxylate on the MoaD subunit of molybdopterin synthase might resemble the ubiquitin-activating step in the ubiquitin-targeted degradation of proteins in eukaryotes. To determine the exact role of MoeB in molybdopterin biosynthesis, the protein was purified after homologous overexpression. Using purified proteins, we have demonstrated the ATP-dependent formation of a complex of MoeB and MoaD adenylate that is stable to gel filtration. Mass spectrometry of the complex revealed a peak of a molecular mass of 9,073 Da, the expected mass of MoaD adenylate. However, unlike the ubiquitin activation reaction, the formation of a thioester intermediate between MoeB and MoaD could not be observed. There was also no evidence for a MoeB-bound sulfur during the sulfuration of MoaD. Amino acid substitutions were generated in every cysteine residue in MoeB. All of these exhibited activity comparable to the wild type, with the exception of mutations in cysteine residues located in putative Zn-binding motifs. For these cysteines, loss of activity correlated with loss of metal binding.     

Opal suppressor phosphoserine tRNA gene and pseudogene are located on human chromosomes 19 and 22, respectively

An opal suppressor phosphoserine tRNA gene and pseudogene have been isolated from a human DNA library and sequenced (O'Neill, V., Eden, F., Pratt, K., and Hatfield, D. (1985) J. Biol. Chem. 260, 2501-2508). Southern hybridization of human genomic DNA with an opal suppressor tRNA probe suggested that the gene and pseudogene are present in single copy. In this study, we have determined the chromosome location of the human gene and pseudogene by utilizing a 193-base pair fragment encoding the opal suppressor phosphoserine tRNA gene as probe to examine DNAs isolated from human-rodent somatic cell hybrids that have segregated human chromosomes. These studies show that the probe hybridized with two regions in the human genome; one is located on chromosome 19 and the second on chromosome 22. By comparing the restriction sites within these two regions to those previously determined for the human opal suppressor phosphoserine tRNA gene and pseudogene, we tentatively assigned the gene to chromosome 19 and the pseudogene to chromosome 22. These assignments were confirmed by utilizing a 350-base pair fragment which was isolated from the 5'-flanking region of the human gene as probe. This fragment hybridized only to chromosome 19, demonstrating unequivocally that the opal suppressor phosphoserine tRNA gene is located on chromosome 19. The flanking probe hybridized to a single homologous band in hamster and in mouse DNA to which the gene probe also hybridized, demonstrating that the 5'-flanking region of the opal suppressor tRNA gene is conserved in mammals. Restriction analysis of DNAs obtained from the white blood cells of 10 separate individuals demonstrates that the gene is polymorphic. This study provides two additional markers for the human genome and constitutes only the second set of two tRNA genes assigned to human chromosomes.     

Mycobacterium tuberculosis CwsA Interacts with CrgA and Wag31, and the CrgA-CwsA Complex Is Involved in Peptidoglycan Synthesis and Cell Shape Determination

Bacterial cell division and cell wall synthesis are highly coordinated processes involving multiple proteins. Here, we show that Rv0008c, a novel small membrane protein from Mycobacterium tuberculosis, localizes to the poles and on membranes and shows an overall punctate localization throughout the cell. Furthermore, Rv0008c interacts with two proteins, CrgA and Wag31, implicated in peptidoglycan (PG) synthesis in mycobacteria. Deletion of the Rv0008c homolog in M. smegmatis, MSMEG_0023, caused bulged cell poles, formation of rounded cells, and defects in polar localization of Wag31 and cell wall synthesis, with cell wall synthesis measured by the incorporation of the [¹⁴C]N-acetylglucosamine cell wall precursor. The M. smegmatis MSMEG_0023 crgA double mutant strain showed severe defects in growth, viability, cell wall synthesis, cell shape, and the localization of the FtsZ, FtsI, and Wag31 proteins. The double mutant strain also exhibited increased autolytic activity in the presence of detergents. Because CrgA and Wag31 proteins interact with FtsI individually, we believe that regulated cell wall synthesis and cell shape maintenance require the concerted actions of the CrgA, Rv0008c, FtsI, and Wag31 proteins. We propose that, together, CrgA and Rv0008c, renamed CwsA for cell wall synthesis and cell shape protein A, play crucial roles in septal and polar PG synthesis and help coordinate these processes with the FtsZ-ring assembly in mycobacteria.     

GQ-16, a Novel Peroxisome Proliferator-activated Receptor γ (PPARγ) Ligand, Promotes Insulin Sensitization without Weight Gain

The recent discovery that peroxisome proliferator-activated receptor γ (PPARγ) targeted anti-diabetic drugs function by inhibiting Cdk5-mediated phosphorylation of the receptor has provided a new viewpoint to evaluate and perhaps develop improved insulin-sensitizing agents. Herein we report the development of a novel thiazolidinedione that retains similar anti-diabetic efficacy as rosiglitazone in mice yet does not elicit weight gain or edema, common side effects associated with full PPARγ activation. Further characterization of this compound shows GQ-16 to be an effective inhibitor of Cdk5-mediated phosphorylation of PPARγ. The structure of GQ-16 bound to PPARγ demonstrates that the compound utilizes a binding mode distinct from other reported PPARγ ligands, although it does share some structural features with other partial agonists, such as MRL-24 and PA-082, that have similarly been reported to dissociate insulin sensitization from weight gain. Hydrogen/deuterium exchange studies reveal that GQ-16 strongly stabilizes the β-sheet region of the receptor, presumably explaining the compound's efficacy in inhibiting Cdk5-mediated phosphorylation of Ser-273. Molecular dynamics simulations suggest that the partial agonist activity of GQ-16 results from the compound's weak ability to stabilize helix 12 in its active conformation. Our results suggest that the emerging model, whereby "ideal" PPARγ-based therapeutics stabilize the β-sheet/Ser-273 region and inhibit Cdk5-mediated phosphorylation while minimally invoking adipogenesis and classical agonism, is indeed a valid framework to develop improved PPARγ modulators that retain antidiabetic actions while minimizing untoward effects.     

The RGSGR amino acid motif of the intercellular signalling protein, HetN, is required for patterning of heterocysts in Anabaena sp. strain PCC 7120

Nitrogen-fixing heterocysts are arranged in a periodic pattern on filaments of the cyanobacterium Anabaena sp. strain PCC 7120 under conditions of limiting combined nitrogen. Patterning requires two inhibitors of heterocyst differentiation, PatS and HetN, which work at different stages of differentiation by laterally suppressing levels of an activator of differentiation, HetR, in cells adjacent to source cells. Here we show that the RGSGR sequence in the 287-amino-acid HetN protein, which is shared by PatS, is critical for patterning. Conservative substitutions in any of the five amino acids lowered the extent to which HetN inhibited differentiation when overproduced and altered the pattern of heterocysts in filaments with an otherwise wild-type genetic background. Conversely, substitution of amino acids comprising the putative catalytic triad of this predicted reductase had no effect on inhibition or patterning. Deletion of putative domains of HetN suggested that the RGSGR motif is the primary component of HetN required for both its inhibitory and patterning activity, and that localization to the cell envelope is not required for patterning of heterocysts. The intercellular signalling proteins PatS and HetN use the same amino acid motif to regulate different stages of heterocyst patterning.     

The G473D mutation impairs dimerization and catalysis in human sulfite oxidase

Among the mutations identified in patients with isolated sulfite oxidase deficiency, the G473D variant is of particular interest since sedimentation analysis reveals that this variant is a monomer, and the importance of the wild-type dimeric state of mammalian sulfite oxidase is not yet well understood. Analysis of recombinant G473D sulfite oxidase indicated that it is severely impaired both in the ability to bind sulfite and in catalysis, with a second-order rate constant 5 orders of magnitude lower than that of the wild type. To elucidate the specific reasons for the severe effects seen in the G473D variant, several other variants were created, including G473A, G473W, and the double mutant R212A/G473D. Despite the inability to form a stable dimer, the G473W variant had 5-fold higher activity than G473D and nearly wild-type activity at pH 7.0 when ferricyanide was the electron acceptor. In contrast, the R212A/G473D variant demonstrated some ability to oligomerize but had undetectable activity. The G473A variant retained the ability to dimerize and had steady-state activity that was comparable to that of the wild type. Furthermore, stopped-flow analysis of the reductive half-reaction of this variant yielded a rate constant nearly 3 times higher than that of the wild type. Examination of the secondary structures of the variants by CD spectroscopy indicated significant random-coil formation in G473D, G473W, and R212A/G473D. These results demonstrate that both the charge and the large size of an Asp residue in this position contribute to the severe effects seen in a patient with the G473D mutation, by causing partial misfolding and monomerization of sulfite oxidase and attenuating both substrate binding and catalytic efficiency during the reaction cycle.