Search

Clinicians often report that their tried-and-true therapies do not seem to be working as well as they have in the past. In my own nutritional practice, there-was always a handful [...]

Reactive oxygen intermediates generated by neutrophils kill bacteria and are implicated in inflammatory tissue injury, but precise molecular targets are undefined. We demonstrate that neutrophils use myeloperoxidase (MPO) to convert methionine residues of ingested Escherichia coli to methionine sulfoxide in high yield. Neutrophils deficient in individual components of the MPO system (MPO, H202, chloride) exhibited impaired bactericidal activity and impaired capacity to oxidize methionine. HOCI, the principal physiologic product of the MPO system, is a highly efficient oxidant for methionine, and its microbicidal effects were found to correspond linearly with oxidation of methionine residues in bacterial cytosolic and inner membrane proteins. In contrast, outer envelope proteins were initially oxidized without associated microbicidal effect. Disruption of bacterial methionine sulfoxide repair systems rendered E. coli more susceptible to killing by HOCI, whereas over-expression of a repair enzyme, methionine sulfoxide reductase A, rendered them resistant, suggesting a direct role for methionine oxidation in bactericidal activity. Prominent among oxidized bacterial proteins were those engaged in synthesis and translocation of peptides to the cell envelope, an essential physiological function. Moreover, HOCI impaired protein translocation early in the course of bacterial killing. Together, our findings indicate that MPO-mediated methionine oxidation contributes to bacterial killing by neutrophils. The findings further suggest that protein translocation to the cell envelope is one important pathway targeted for damage. bactericidal | microbicidal www.pnas.org/cgi/doi/10.1073/pnas.0909464106

Cobalamin-dependent methionine synthase (MetH) is a modular protein that catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine to produce methionine and tetrahydrofolate. The cobalamin cofactor, which serves as both acceptor and donor of the methyl group, is oxidized once every [approximately equal to]2,000 catalytic cycles and must be reactivated by the uptake of an electron from reduced flavodoxin and a methyl group from S-adenosyl-L-methionine (AdoMet). Previous structures of a C-terminal fragment of MetH (Met[H.sup.CT]) revealed a reactivation conformation that juxtaposes the cobalamin- and AdoMet-binding domains. Here we describe 2 structures of a disulfide stabilized Met[H.sup.CT] ([sub.s-s]Met[H.SUP.CT]) that offer further insight into the reactivation of MetH. The structure of [sub.s-s]Met[H.sup.CT] with cob(II)alamin and S-adenosyl-L-homocysteine represents the enzyme in the reactivation step preceding electron transfer from flavodoxin. The structure supports earlier suggestions that the enzyme acts to lower the reduction potential of the Co(II)/Co(I) couple by elongating the bond between the cobalt and its upper axial water ligand, effectively making the cobalt 4-coordinate, and illuminates the role of Tyr-1139 in the stabilization of this 4-coordinate state. The structure of [sub.s-s]Met[H.SUP.CT] with aquocobalamin may represent a transient state at the end of reactivation as the newly remethylated 5-coordinate methylcobalamin returns to the 6-coordinate state, triggering the rearrangement to a catalytic conformation. corrinoid methyltransferase | multirnodular protein | protein conformation | cob(II)alamin coordination | enzyme catalysis www.pnas.org/cgi/doi/10.1073/pnas.0906132106

Impaired transfer of methyl groups via the methionine cycle leads to plasma hyperhomocysteinemia. The tissue sources of plasma homocysteine in vivo have not been quantified nor whether hyperhomocysteinemia is due to increased entry or decreased removal. These issues were addressed in female rats offered diets with either adequate or excess methionine (additional methyl groups) with or without folate and choline (impaired methyl group transfer) for 5 wk. Whole body and tissue metabolism was measured based on isotopomer analysis following infusion with either [1-[sup.13]C,methyl-[sup.2][H.sub.3]]methionine or [U-[sup.13]C]methionine plus [1-[sup.13]C]homocysteine. Although the fraction of intracellular methionine derived from methylation of homocysteine was highest in liver (0.18-0.21), most was retained. In contrast, the pancreas exported to plasma more of methionine synthesized de novo. The pancreas also exported homocysteine to plasma, and this matched the contribution from liver. Synthesis of methionine from homocysteine was reduced in most tissues with excess methionine supply and was also lowered in fiver (P < 0.01) with diets devoid of folate and choline. Plasma homocysteine concentration (P < 0.001) and flux (P = 0.001) increased with folate plus choline deficiency, although the latter still represented < 12% of estimated tissue production. Hyperhomocysteinemia also increased (P < 0.01) the inflow of homocysteine into most tissues, including heart. These findings indicate that a full understanding of hyperhomocysteinemia needs to include metabolism in a variety of organs, rather than an exclusive focus on the liver. Furthermore, the high influx of homocysteine into cardiac tissue may relate to the known association between fiomocysteinemia and hypertension. stable isotopes; isotopomer analysis; homocysteine methylation; metabolic models

The transport systems involved in intestinal methionine (Met) absorption are described as [Na.sup.+]-dependent and [Na.sup.+]-independent mechanisms. However, since recent studies have suggested the importance of the [H.sup.+] gradient as a driving force for intestinal nutrient absorption, the aim of the present work was to test whether Met transport across the apical membrane of Caco-2 cells is affected by extracellular pH. The results show that Land D-Met uptake was increased by lowering extracellular pH from 7.4 to 5.5, in both the presence and absence of [Na.sup.+]. Cis-inhibition experiments revealed that inhibition of L-Met transport by 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) or L-lysine (L-Lys) was higher at a pH of 5.5. Moreover, the BCH-insensitive component was not affected by pH, whereas the L-Lys-insensitive component was increased by lowering extracellular pH, thus suggesting the participation of system L. The contribution of another mechanism, sensitive to both BCH and L-Lys, was also considered. The inhibition obtained with taurine (Tau) was also higher at a pH of 5.5, thus suggesting the involvement of system [B.sup.0,+] on pH-stimulated component. As for D-Met uptake, the results showed higher inhibition with L-Lys and Tau at a pH of 5.5 and no effect on the L-Lys- or Tau-insensitive component. In conclusion, Met transport across the apical membrane of Caco-2 cells is increased by low extracellular pH as the result of the stimulation of two transport systems functionally identified with systems L and [B.sup.0,+] for L-Met and with system [B.sup.0,+] for D-Met. intestine; amino acid transport; [H.sup.+] gradient

Membrane proteins represent formidable challenges for many analytical techniques. Studies on these systems are often carried out after surfactant solubilization. Unfortunately, such a non-natural protein environment can affect conformation and stability, and it offers only partial protection against aggregation. This work employs bacteriorhodopsin (BR) as a model system for in situ structural studies on a membrane protein in its natural lipid bilayer. BR-containing purple membrane suspensions were exposed to hydroxyl radicals, generated by nanosecond laser photolysis of dilute aqueous [H.sub.2][O.sub.2]. The experiments rely on the premise that oxidative labeling occurs mainly at solvent-exposed side chains, whereas sites that are sterically protected will react to a much lesser extent. Following x OH exposure, the protein was analyzed by tryptic peptide mapping and electrospray tandem mass spectrometry. Oxidative labeling of BR was found to occur only at its nine Met residues. This is in contrast to the behavior of previously studied water-soluble proteins, which generally undergo modifications at many different types of residues. In those earlier experiments the high reactivity of Met has hampered its use as a structural probe. In contrast, the Met oxidation pattern observed here is in excellent agreement with the native BR structure. Extensive labeling is seen for Met32, 68, and 163, all of which are located in solvent-exposed loops. The remaining six Met residues are deeply buried and show severalfold less oxidation. Our results demonstrate the usefulness of Met oxidative labeling for structural studies on membrane proteins, especially when considering that many of these species are methionine-rich. The introduction of additional Met residues as conformational probes, as well as in vivo structural investigations, represents exciting future extensions of the methodology described here.

Methionine is an essential amino acid that is limiting in maize-(Zea mays L.) based diets. The objective of this work was to determine whether we could alter grain methionine concentration in random-mated maize populations by mass selection for methionine concentration using a microbial assay. In one study, we developed two populations by selecting for high or low methionine concentration (HM or LM, respectively) for three generations starting from the random-mated population BS11. Grain from these populations was used to formulate diets for a feeding trial in which 15 rats were fed HM grain and 15 rats were fed LM grain. Rats on the HM diet had a 0.018 higher feed efficiency (g gain/g feed) than rats on the LM diet. In a second study, we performed three cycles of selection for high or low methionine concentration starting with two random-mated populations, BS11 and BS31. We evaluated each cycle of selection in a field trial with two replications in each of two years. Methionine concentration was significantly correlated with the cycle of selection, changing on average 0.004 g methionine/100 g grain per cycle. Kernel mass, %N, oil, protein, starch, tryptophan, and lysine concentration did not exhibit significant correlations with cycle of selection. We conclude that recurrent selection for grain methionine concentration using a microbial assay is an effective method to alter methionine content.

[B.sub.12]-dependent methionine synthase (MetH) from Escherichia coli is a large modular protein that is alternately methylated by methyltetrahydrofolate to form methylcobalamin and demethylated by homocysteine to form cob(l)alamin. Major domain rearrangements are required to allow cobalamin to react with three different substrates: homocysteine, methyltetrahydrofolate, and S-adenosyl-L-methionine (AdoMet). These same rearrangements appear to preclude crystallization of the wild-type enzyme. Disulfide cross-linking was used to lock a C-terminal fragment of the enzyme into a unique conformation. Cysteine point mutations were introduced at Ile-690 and Gly-743. These cysteine residues span the cap and the cobalamin-binding module and form a cross-link that reduces the conformational space accessed by the enzyme, facilitating protein crystallization. Here, we describe an x-ray structure of the mutant fragment in the reactivation conformation; this conformation enables the transfer of a methyl group from AdoMet to the cobalamin cofactor. In the structure, the axial ligand to the cobalamin, His-759, dissociates from the cobalamin and forms intermodular contacts with residues in the AdoMet-binding module. This unanticipated intermodular interaction is expected to play a major role in controlling the distribution of conformers required for the catalytic and the reactivation cycles of the enzyme. multimodular protein | protein conformation | vitamin [B.sub.12]

Enzymes possessing catalytic zinc centers perform a variety of fundamental processes in nature, including methyl transfer to thiols. Cobalamin-independent (MetE) and cobalamin-dependent (MetH) methionine synthases are two such enzyme families. Although they perform the same net reaction, transfer of a methyl group from methyltetrahydrofolate to homocysteine (Hcy) to form methionine, they display markedly different catalytic strategies, modular organization, and active site zinc centers. Here we report crystal structures of zinc-replete Mete and MetH, both in the presence and absence of Hcy. Structural investigation of the catalytic zinc sites of these two methyltransferases reveals an unexpected inversion of zinc geometry upon binding of Hcy and displacement of an endogenous ligand in both enzymes. In both cases a significant movement of the zinc relative to the protein scaffold accompanies inversion. These structures provide new information on the activation of thiols by zinc-containing enzymes and have led us to propose a paradigm for the mechanism of action of the catalytic zinc sites in these and related methyltransferases. Specifically, zinc is mobile in the active sites of MetE and MetH, and its dynamic nature helps facilitate the active site conformational changes necessary for thiol activation and methyl transfer. cobalamin-dependent methionine synthase | cobalamin-independent methionine synthase | methyl transferases | zinc enzymes | zinc inversion

This laboratory and others have shown that agents that inhibit the in vitro catalytic activity of methionine aminopeptidase-2 (MetAP2) are effective in blocking angiogenesis and tumor growth in preclinical models. However, these prototype MetAP2 inhibitors are clearly not optimized for therapeutic use in the clinic. We have discovered an orally active class of MetAP2 inhibitors, the anthranilic acid sulfonamides exemplified by A-800141, which is highly specific for MetAP2. This orally bioavailable inhibitor exhibits an antiangiogenesis effect and a broad anticancer activity in a variety of tumor xenografts including B cell lymphoma, neuroblastoma, and prostate and colon carcinomas, either as a single agent or in combination with cytotoxic agents. We also have developed a biomarker assay to evaluate in vivo MetAP2 inhibition in circulating mononuclear cells and in tumors. This biomarker assay is based on the N-terminal methionine status of the MetAP2-specific substrate GAPDH in these cells. In cell cultures in vitro, the sulfonamide MetAP2 inhibitor A-800141 caused the formation of GAPDH variants with an unprocessed N-terminal methionine. A-800141 blocked tumor growth and MetAP2 activity in a similar dose-response in mouse models, demonstrating the antitumor effects seen for A-800141 are causally connected to MetAP2 inhibition in vivo. The sulfonamide MetAP2 inhibitor and GAPDH biomarker in circulating leukocytes may be used for the development of a cancer treatment. angiogenesis | biomarker | cancer therapy | GAPDH | MetAP2

Homocysteine (Hcy) and S-adenosylhomocysteine (AdoHcy) are critical intermediates of methionine metabolism. To investigate which, if either, of these compounds is more closely related to atherosclerosis, we fed 5 groups of apolipoprotein E (apoE)-deficient mice different diets for 8 wk to induce changes in their plasma Hcy and AdoHcy concentrations. These included an AIN-93G control diet (C), this C diet supplemented with methionine (M), the M diet deficient in folates, vitamin B-6, and vitamin B-12 (M-V), this M diet supplemented with these 8 vitamins (M+V), and a C diet deficient in B vitamins (C-V). Compared with controls, mice fed the C-V diet had a moderate elevation in their plasma total Hcy (tHcy) levels; however, their plasma AdoHcy concentration and atherosclerotic lesion areas were not different. In contrast, the mice fed the M+V diet had larger atherosclerotic lesion areas and elevated plasma AdoHcy concentrations but their plasma tHcy concentration did not differ from that of the group C mice. The plasma AdoHcy concentration and aortic sinus lesion areas were positively correlated (r = 0.866; P < 0.001). We observed a negative correlation between the plasma AdoHcy concentration and both the DNA methyltransferase activity (r = -0.792; P < 0.001) and global DNA methylation status (r = -0.824; P < 0.001) in the aortic tissue. Hence, our study suggests that plasma AdoHcy is a better biomarker of atherosclerosis than Hcy and may accelerate the development of atheroscierotic lesions in apoE-deficient mice that have been fed a high methionine diet. The mechanisms underlying this effect may be related to the AdoHcymediated inhibition of DNA methylation in the aortic tissue.

Nonalcoholic fatty liver disease (NAFLD) has become common liver disease in Western countries. There is accumulating evidence that mitochondria play a key role in NAFLD. Nevertheless, the mitochondrial consequences of steatohepatitis are still unknown. The bioenergetic changes induced in a methionine- and choline-deficient diet (MCDD) model of steatohepatitis were studied in rats. Liver mitochondria from MCDD rats exhibited a higher rate of oxidative phosphorylation with various substrates, a rise in cytochrome oxidase (COX) activity, and an increased content in cytochrome a[a.sub.3]. This higher oxidative activity was associated with a low efficiency of the oxidative phosphorylation (ATP/O, i.e., number of ATP synthesized/ natom O consumed). Addition of a low concentration of cyanide, a specific COX inhibitor, restored the efficiency of mitochondria from MCDD rats back to the control level. Furthermore, the relation between respiratory rate and protonmotive force (in the nonphosphorylating state) was shifted to the left in mitochondria from MCDD rats, with or without cyanide. These results indicated that, in MCDD rats, mitochondrial ATP synthesis efficiency was decreased in relation to both proton pump slipping at the COX level and increased proton leak although the relative contribution of each phenomenon could not be discriminated. MCDD mitochondria also showed a low reactive oxygen species production and a high lipid oxidation potential. We conclude that, in MCDD-fed rats, liver mitochondria exhibit an energy wastage that may contribute to limit steatosis and oxidative stress in this model of steatohepatitis. nonalcoholic steatohepatitis; mitochondria; uncoupling

In 2 experiments, 6 ruminally cannulated Holstein steers (205 [+ or -] 23 and 161 [+ or -] 14 kg initial BW in Exp. 1 and 2, respectively) housed in metabolism crates were used in 6 x 6 Latin squares to study the effects of excess AA supply on Met (Exp. 1) and Leu (Exp. 2) use. All steers received a diet based on soybean hulls (DMI = 2.66 and 2.45 kg/d in Exp. 1 and 2, respectively); ruminal infusions of 200 g of acetate/d, 200 g of propionate/d, and 50 g of butyrate/d, as well as abomasal infusion of 300 g of glucose/d to provide energy without increasing the microbial protein supply; and abomasal infusions of a mixture of all essential AA except Met (Exp. 1) or Leu (Exp. 2). Periods were 6 d, with 2-d adaptations and 4 d to collect N balance data. All treatments were abomasally infused. In Exp. 1, treatments were arranged as a 2 x 3 factorial, with 2 amounts of L-Met (0 or 4 g/d) and 3 AA supplements (no additional AA, control; 100 g/d of nonessential AA + 100 g/d of essential AA, NEAA + EAA; and 200 g/d of essential AA, EAA). Supplemental Met increased (P < 0.01) retained N and decreased (P < 0.01) urinary N and urinary urea N. Retained N increased (P < 0.01) with NEAA + EAA only when 4 g/d of Met was provided, but it increased (P < 0.01) with EAA with or without supplemental Met. Both AA treatments increased (P < 0.01) plasma urea and serum insulin. Plasma glucose decreased (P = 0.03) with supplemental Met. In Exp. 2, treatments were arranged as a 2 x 3 factorial with 2 amounts of L-Leu (0 or 4 g/d) and 3 AA supplements (control, NEAA + EAA, and EAA). Supplemental Leu increased (P < 0.01) retained N and decreased (P < 0.01) urinary N and urinary urea N. Both AA treatments increased (P < 0.01) retained N, and they also increased (P < 0.01) urinary N, urinary urea N, and plasma urea. Serum insulin increased (P = 0.06) with supplemental Leu and tended (P = 0.10) to increase with both AA treatments. Supplementation with excess AA improved Met and Leu use for protein deposition by growing cattle. Key words: amino acid, cattle, growth, leucine, methionine, utilization

Three experiments were conducted to determine the true ileal digestible (TID) Lys and sulfur AA (SAA) requirement and to compare the bioefficacy of 2-hydroxy-4-(methylthio)butanoic acid (HMTBA) and DL-MET as Met sources in nursery pigs. Experiment 1 included 2 studies: 1 was 662 nursery pigs (Triumph 4 x PIC C22; initial BW 12.2 [+ or -] 0.18 kg) allotted to 1 of 5 dietary treatments with TID Lys concentrations ranging from 1.10 to 1.50%; and the second study was 665 nursery pigs (Triumph 4 x PIC C22; initial BW 12.3 [+ or -] 0.18 kg) allotted to 1 of 5 dietary treatments with TID SAA concentration ranging from 0.63 to 0.90%. In Exp. 2, 638 nursery pigs (Triumph 4 x PIC C22; initial BW 13.0 [+ or -] 0.16 kg) were allotted to the same 5 SAA dietary treatments as in Exp. 1. In Exp. 3, 1,232 pigs (Triumph 4 x PIC C22; initial BW 11.0 [+ or -] 0.30 kg) were allotted to 1 of 7 dietary treatments. The basal diet (diet 1) was supplemented with high concentrations of synthetic AA but no Met; this resulted in a dietary concentration of TID Lys of 1.30% and TID SAA of 0.50%. Diets 2 to 7 were the basal diet supplemented with 3 equimolar levels of HMTBA or DL-MET to provide TID SAA concentrations of 0.56, 0.62, and 0.68%, respectively. In Exp. 1, increasing TID Lys from 1.10 to 1.50% increased ADG (quadratic; P < 0.05) and improved G:F (linear; P < 0.002). The pooled data of Exp. 1 (SAA study) and Exp. 2 indicated that increasing TID SAA from 0.63 to 0.90% increased ADG (quadratic; P < 0.01) and improved G:F (quadratic; P < 0.01). Various methods of analyzing the growth response surface indicated that the optimal TID Lys concentration ranged from 1.28 to 1.32% for ADG (Exp. 1), and the optimal TID SAA concentration ranged from 0.73 to 0.77% for ADG and 0.80 to 0.83% for G:F (pooled Exp. 1 and 2), respectively. In Exp. 3, increasing TID SAA concentrations from 0.50 to 0.68% resulted in a linear improvement of ADG (P < 0.001), ADFI (P < 0.05), and G:F (P < 0.001). The best fit comparison of HMTBA and DLMET was determined by the Schwartz Bayesian Information Criteria index, which indicated the average relative efficacy of HMTBA vs. DL-MET was 111%, with 95% confidence interval of 83 to 138%, within the range of TID SAA tested. Thus, the TID Lys and SAA requirements of modern lean-genotype pigs from 11- to 26-kg were greater than the 1998 NRC recommendations, and both HMTBA and DL-MET as Met sources can supply equimolar amounts of Met activity. Key words: DL-methionine, growth, 2-hydroxy-4-(methylthio)butanoic acid, methionine bioefficacy, pig, sulfur amino acid

Two experiments were conducted to evaluate the efficacy of dietary DL-methionine hydroxy analog-free acid (MHA-FA, 88%) compared with DL-methionine (DLM, 99%) as Met sources in pigs. In Exp. 1, a total of 245 crossbred pigs (initial BW of 6.4 kg [SD = 0.5]) were allotted to 7 treatments in 7 replicates for an experimental period of 28 d. The basal diet (BD)was formulated to contain 17.5% CP and 0.21% Met. Dietary treatments included 1) BD, 2) BD + 0.030% DLM, 3) BD + 0.060% DLM, 4) BD + 0.090% DLM, 5) BD + 0.034% MHA-FA, 6) BD + 0.068% MHA-FA, and 7) BD + 0.103% MHA-FA; the MHA-FA was supplemented on an equimolar basis to the DLM. Because of a nonlinear response, exponential regression analysis was used to evaluate the responses, and a comparison of the equations was then made to determine the relative effectiveness of the 2 Met sources. With increases in dietary Met, weight gain increased (P < 0.05). Compared with DLM on a product-to-product (wt/wt) basis, the relative effectiveness of MHA-FA was calculated to be 73% for increasing weight gain and 54% for decreasing the feed:gain. In Exp. 2, a total of 30 weanling barrows [initial BW of 16.8 kg(SD = 2.8)] were used in a metabolism study to evaluate the relative value of MHA-FA to DLM. The BD was formulated to contain 16.9% CP and 0.21% Met. Dietary treatments included 1) BD, 2) BD + 0.030% DLM, 3) BD + 0.060% DLM, 4) BD + 0.046% MHA-FA, and 5) BD + 0.092% MHA-FA; the MHA-FA levels were chosen based on a pre-experiment estimate of bioequivalence in an attempt to provide approximately equal pig responses. There was no difference in fecal N output among the treatments; however, urine N linearly decreased with increasing concentrations of both sources (P = 0.034 for DLM, and P = 0.007 for MHA-FA), which resulted in a linear increase in retained N for both DLM (P = 0.012) and MHA-FA (P = 0.005). In addition, N retention (% of intake) linearly increased with increasing level of DLM (P = 0.014) and MHA-FA (P = 0.007). Using a slope-ratio procedure for comparison of the responses from the 2 sources, the relative biological equivalence value of MHA-FA to DLM in this experiment was 64.2% based on percent N retention and 66.3% based on the grams of N retained per day. Based on the results from both experiments, these data indicated that the mean relative bioequivalence of MHA-FA to DLM was 64% on a product-to-product (wt/wt) basis or 73% on an equimolar basis. Key words: bioefficacy, methionine, methionine hydroxy analog, pig

A study was conducted to analyze the structural and fibrillation properties of a series of alpha-synuclein mutants. The result shows that the degree of inhibition of fibrillation by MetO alpha-synuclein is proportional to the number of oxidized methionines.