Effect of methionine and its related compounds on rumen bacterial activity

The effect of several methionine sources (L‐methionine = L‐MET; DL‐ methionine = DL‐MET, DL‐S‐methyl‐methionine‐sulphonium‐chloride = SMM; N‐hydroxymethyl‐DL‐methionine‐Ca = NHM; methionine‐hydroxy‐analog free acid=MHA; methionine‐sulphoxyde=MSO) on rumen bacterial growth was studied using a new methodical approach which utilises a methionine free assay medium (Bacto Methionine Assay Media, Difco) supplemented by increasing quantities of the methionine sources and inoculated with one drop of diluted rumen bacteria. The optical density was measured after 18 h incubation on 39 °C.

L‐ and DL‐MET promoted the highest growth response, while SMM and NHM exerted significantly (p < 0.05) lower optical densities. MHA and MSO showed no growth response.

The methodical approach and the possible bacterial strains, which might have contributed to the growth response have been discussed.     

Structural insights into the catalytic mechanism of 5-methylthioribose 1-phosphate isomerase

5-Methylthioribose 1-phosphate isomerase (M1Pi) is a crucial enzyme involved in the universally conserved methionine salvage pathway (MSP) where it is known to catalyze the conversion of 5-methylthioribose 1-phosphate (MTR-1-P) to 5-methylthioribulose 1-phosphate (MTRu-1-P) via a mechanism which remains unspecified till date. Furthermore, although M1Pi has a discrete function, it surprisingly shares high structural similarity with two functionally non-related proteins such as ribose-1,5-bisphosphate isomerase (R15Pi) and the regulatory subunits of eukaryotic translation initiation factor 2B (eIF2B). To identify the distinct structural features that lead to divergent functional obligations of M1Pi as well as to understand the mechanism of enzyme catalysis, the crystal structure of M1Pi from a hyperthermophilic archaeon Pyrococcus horikoshii OT3 was determined. A meticulous structural investigation of the dimeric M1Pi revealed the presence of an N-terminal extension and a hydrophobic patch absent in R15Pi and the regulatory α-subunit of eIF2B. Furthermore, unlike R15Pi in which a kink formation is observed in one of the helices, the domain movement of M1Pi is distinguished by a forward shift in a loop covering the active-site pocket. All these structural attributes contribute towards a hydrophobic microenvironment in the vicinity of the active site of the enzyme making it favorable for the reaction mechanism to commence. Thus, a hydrophobic active-site microenvironment in addition to the availability of optimal amino-acid residues surrounding the catalytic residues in M1Pi led us to propose its probable reaction mechanism via a cis-phosphoenolate intermediate formation.     

Functional amino acid supplementation postweaning mitigates the response of normal birth weight more than for low birth weight pigs to a subsequent Salmonella challenge

Previous work has shown that dietary supplementation with key functional amino acids (FAA) improves growth performance and immune status of disease-challenged normal birth weight (NBW) pigs. It is not known whether FAA supplementation attenuates the effects of a subsequent disease challenge or whether this response is similar in low birth weight (LBW) pigs. The objective was to determine the effects of birth weight and FAA supplementation during the postweaning period in Salmonella-challenged pigs. Thirty-two LBW (1.08 ± 0.11 kg) and NBW (1.58 ± 0.11 kg) pigs were assigned to a nursery feeding program at weaning (25 d) for 31 days in a 2 × 2 factorial arrangement. Factors were birth weight category (LBW vs. NBW) and basal (FAA–) or supplemented FAA profile (FAA+; Thr, Met, and Trp at 120% of requirements). At d 31, pigs were placed onto a common grower diet and, after a 7-d adaptation period, were inoculated with Salmonella Typhimurium (ST; 2.2 × 10⁹ colony-forming units/mL) and monitored for 7-d postinoculation. Growth performance, rectal temperature, fecal score, indicators of gut health, ST shedding score in feces, intestinal ST colonization and translocation, and blood parameters of acute-phase response and antioxidant balance were measured pre- and postinoculation. Inoculation with ST increased temperature and fecal score, and the overall rectal temperature was higher in LBW compared to NBW pigs (P < 0.05). Postinoculation (d 7), reduced:oxidized glutathione was increased in NBW compared to LBW pigs (P < 0.05). Salmonella shedding and translocation to spleen were lower in NBW-FAA+ compared to NBW-FAA? pigs (P < 0.05). Postinoculation average daily gain was higher in NBW-FAA+ (P < 0.05) compared to the other groups. Postinoculation haptoglobin, superoxide dismutase, and colonic myeloperoxidase were increased in LBW-FAA? pigs (P < 0.05). Ileal alkaline phosphatase was decreased in LBW compared to NBW (P < 0.05). Overall, FAA supplementation represents a potential strategy to mitigate the effect of enteric disease challenge in NBW, but not LBW pigs.     

NMR assignments of 1H, 13C and 15N spectra of methionine sulfoxide reductase B1 from Mus musculus

Isotopically labeled, ¹⁵N and ¹⁵N/¹³C forms of recombinant methionine-r-sulfoxide reductase 1 (MsrB1, SelR) from Mus musculus were produced, in which catalytic selenocysteine was replaced with cysteine. We report here the ¹H, ¹⁵N and ¹³C NMR assignment of the reduced form of this mammalian protein.     

The central nervous system of Bulla gouldiana: peptide localization

In the present study, we describe the structure of the central nervous system (CNS) of the marine gastropod Bulla gouldiana, and compare it with the structure of the CNS of the related mollusc, Aplysia californica. In addition, we performed an immunohistochemical analysis of a series of peptides, and the synaptic vesicle protein, synapsin I, in the central nervous system of B. gouldiana. The most common peptide in the B. gouldiana nervous system is the molluscan cardioexcitatory peptide (FMRFamide), which is present in a significant proportion of B. gouldiana neurons. A smaller number of neurons exhibit immunoreactivity to antisera raised against the calcitonin gene related peptide, vasopressin, vasoactive intestinal peptide, cholecystokinin, galanin and enkephalin. In some instances there is colocalization of two or more peptides. Very few neurons or axons exhibit synapsin I-like immunoreactivity. The patterns of immunoreactivity to these antisera is quite similar to the patterns that have been described in other gastropods, including Lymnaea stagnalis and Aplysia californica. These observations emphasize the importance of FMRFamide-like compounds in phylogenetically old nervous systems and indicate that compounds similar to mammalian peptides are present in the gastropod. Thus, the production of a wide variety of peptide molecules and their use in neuronal function appears to be a highly conserved phylogenetic process.     

Signal sequence recognition and protein targeting

Intracellular traffic is often controlled not by highways, but by handshakes and partner introductions within a cellular network. Recently determined structures suggest how signal sequences are recognized and how the GTP affinities of the signal recognition particle and its receptor are coupled to the targeting of ribosomes to translocational membrane pores. The structure of signal peptidase suggests how it releases functional proteins.     

Specificity of the dynorphin-processing endoprotease: comparison with prohormone convertases

The cleavage specificity of a monobasic processing dynorphin converting endoprotease is examined with a series of quench fluorescent peptide substrates and compared with the cleavage specificity of prohormone convertases. A dynorphin B-29-derived peptide, Abz-Arg-Arg-Gln-Phe-Lys-Val-Val-Thr-Arg-Ser-Glneddnp (where Abz is o-aminobenzoyl and eddnp is ethylenediamine 2,4-dinitrophenyl), that contains both dibasic and monobasic cleavage sites is efficiently cleaved by the dynorphin converting enzyme and not cleaved by two propeptide processing enzymes, furin and prohormone convertase 1. A shorter prorenin-related peptide, Dnp-Arg-Met-Ala-Arg-Leu-Thr-Leu-eddnp, that contains a monobasic cleavage site is cleaved by the dynorphin converting enzyme and prohormone convertase 1 and not by furin. Substitution of the P1' position by Ala moderately affects cleavage by the dynorphin-processing enzyme and prohormone convertase 1. It is interesting that this substitution results in efficient cleavage by furin. The site of cleavage, as determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry, is N-terminal to the Arg at the P1 position for the dynorphin converting enzyme and C-terminal to the Arg at the P1 position for furin and prohormone convertase 1. Peptides with additional basic residues at the P2 and at P4 positions also serve as substrates for the dynorphin converting enzyme. This enzyme cleaves shorter peptide substrates with significantly lower efficiency as compared with the longer peptide substrates, suggesting that the dynorphin converting enzyme prefers longer peptides that contain monobasic processing sites as substrates. Taken together, these results suggest that the cleavage specificity of the dynorphin converting enzyme is distinct but related to the cleavage specificity of the prohormone convertases and that multiple enzymes could be involved in the processing of peptide hormones and neuropeptides at monobasic and dibasic sites.     

A transporter of<Emphasis Type="Italic"> Escherichia coli</Emphasis> specific for<Emphasis Type="SmallCaps"> l</Emphasis>- and<Emphasis Type="SmallCaps"> d</Emphasis>-methionine is the prototype for a new family within the ABC superfamily

An ABC-type transporter in Escherichia coli that transports both l- and d-methionine, but not other natural amino acids, was identified. This system is the first functionally characterized member of a novel family of bacterial permeases within the ABC superfamily. This family was designated the methionine uptake transporter (MUT) family (TC #3.A.1.23). The proteins that comprise the transporters of this family were analyzed phylogenetically, revealing the probable existence of several sequence-divergent primordial paralogues, no more than two of which have been transmitted to any currently sequenced organism. In addition, MetJ, the pleiotropic methionine repressor protein, was shown to negatively control expression of the operon encoding the ABC-type methionine uptake system. The identification of MetJ binding sites (in gram-negative bacteria) or S-boxes (in gram-positive bacteria) in the promoter regions of several MUT transporter-encoding operons suggests that many MUT family members transport organic sulfur compounds. Electronic Supplementary Material Supplementary material is available for this article if you access the article at . A link in the frame on the left on that page takes you directly to the supplementary material.     

New membrane-associated and soluble peptide methionine sulfoxide reductases in Escherichia coli

It is known that reactive oxygen species can oxidize methionine residues in proteins in a non-stereospecific manner, and cells have mechanisms to reverse this damage. MsrA and MsrB are members of the methionine sulfoxide family of enzymes that specifically reduce the S and R forms, respectively, of methionine sulfoxide in proteins. However, in Escherichia coli the level of MsrB activity is very low which suggested that there may be other enzymes capable of reducing the R epimer of methionine sulfoxide in proteins. Employing a msrA/B double mutant, a new peptide methionine sulfoxide reductase activity has been found associated with membrane vesicles from E. coli. Both the R and S forms of N-acetylmethionine sulfoxide, D-ala-met(o)-enkephalin and methionine sulfoxide, are reduced by this membrane associated activity. The reaction requires NADPH and may explain, in part, how the R form of methionine sulfoxide in proteins is reduced in E. coli. In addition, a new soluble Msr activity was also detected in the soluble extracts of the double mutant that specifically reduces the S epimer of met(o) in proteins.     

Approaches towards understanding methionine biosynthesis in higher plants

Summary. Plants are able to synthesise all amino acids essential for human and animal nutrition. Because the concentrations of some of these dietary constituents, especially methionine, lysine, and threonine, are often low in edible plant sources, research is being performed to understand the physiological, biochemical, and molecular mechanisms that contribute to their transport, synthesis and accumulation in plants. This knowledge can be used to develop strategies allowing a manipulation of crop plants, eventually improving their nutritional quality. This article is intended to serve two purposes. The first is to provide a brief review on the physiology of methionine synthesis in higher plants. The second is to highlight some recent findings linked to the metabolism of methionine in plants due to its regulatory influence on the aspartate pathway and its implication in plant growth. This information can be used to develop strategies to improve methionine content of plants and to provide crops with a higher nutritional value. Received January 28, 2000 Accepted March 3, 2000