Formation of Lysine from, α, ε-Diaminopimelic Acid Contained in Rumen Bacterial Cell Walls by Rumen Ciliate Protozoa

Cell walls containing α,ε-diaminopimelate-l,7-¹⁴C (DAP) was prepared from Escherichia coli isolated from the rumen. After incubation of ciliates with the cell walls, 22.0% of DAP contained in cell walls of E. coli was converted to lysine and pipecolate. Heat-treated mixed rumen bacteria and heat-treated cell walls of mixed rumen bacteria added to the culture medium of rumen ciliates increased 0.572 and 0.934 μmole/ml of sum of lysine and pipecolate, respectively.

From these results, it is clear that rumen ciliate protozoa can form lysine from DAP contained in the mucopeptide of bacterial cell walls. One of the nutritional significance of inhabitation of ciliates in the rumen was revealed.     

A new robust kinetic assay for DAP epimerase activity

DAP epimerase is the penultimate enzyme in the lysine biosynthesis pathway. The most versatile assay for DAP epimerase catalytic activity employs a coupled DAP epimerase–DAP dehydrogenase enzyme system with a commercial mixture of DAP isomers as substrate. DAP dehydrogenase converts meso-DAP to THDP with concomitant reduction of NADP⁺ to NADPH. We show that at high concentrations, accumulation of NADPH results in inhibition of DAPDH, resulting in spurious kinetic data. A new assay has been developed employing DAP decarboxylase that allows the reliable characterisation of DAP epimerase enzyme kinetics.     

Dimerization of Bacterial Diaminopimelate Epimerase Is Essential for Catalysis

Diaminopimelate (DAP) epimerase is involved in the biosynthesis of meso-DAP and lysine, which are important precursors for the synthesis of peptidoglycan, housekeeping proteins, and virulence factors in bacteria. Accordingly, DAP epimerase is a promising antimicrobial target. Previous studies report that DAP epimerase exists as a monomeric enzyme. However, we show using analytical ultracentrifugation, X-ray crystallography, and enzyme kinetic analyses that DAP epimerase from Escherichia coli exists as a functional dimer in solution and the crystal state. Furthermore, the 2.0-Å X-ray crystal structure of the E. coli DAP epimerase dimer shows for the first time that the enzyme exists in an open, active conformation. The importance of dimerization was subsequently probed by using site-directed mutagenesis to generate a monomeric mutant (Y268A). Our studies show that Y268A is catalytically inactive, thus demonstrating that dimerization of DAP epimerase is essential for catalysis. Molecular dynamics simulations indicate that the DAP epimerase monomer is inherently more flexible than the dimer, suggesting that dimerization optimizes protein dynamics to support function. Our findings offer insight into the development of novel antimicrobial agents targeting the dimeric antibiotic target DAP epimerase.     

Effects of salinomycin and vitamin B(6) on the in vitro synthesis of lysine from the stereoisomers of 2,6-diaminopimelic acid by mixed rumen protozoa,bacteria, and their mixture

An in vitro study was conducted to examine the effects of salinomycin (SL) and vitamin B(6) (pyridoxine hydrochloride) (B(6)) on the production of lysine from the three stereoisomers of 2,6-diaminopimelic acid (DAP-SI) by mixed rumen protozoa (P), mixed rumen bacteria (B), and their mixture (PB). P, B, and PB were isolated from the rumen of goats given a concentrate mixture and lucerne cubes, separately incubated for 12 h with and without DAP-SI (5 mM) as a substrate and SL (5 &mgr;g/ml) and/or B(6) (10 &mgr;g/ml) as additives. In P suspensions, SL and B(6) reduced the amount of DAP-SI by 2.1 times (p<0.001, where p is probability) and 19.9% (p<0.05), respectively, and also increased the production of lysine by 2.4 times (p<0.001) and 26.8% (p <0.05), respectively, during 12 h incubation. In B suspensions, the reductions of DAP-SI with a single addition of SL or B(6) were 8.5% (p<0.001) and 2.7%, respectively, and lysine production increased by 54.3 and 32.9% (p<0.001), respectively, during 12 h incubation. In PB suspensions, the reductions of DAP-SI were 21.9 and 11.7% (p<0.001) with a single addition of SL or B(6), respectively, and the production of lysine increased by 81.4 and 39.4% (p<0.001), respectively, during 12 h incubation. When SL and B(6) were added together to the P, B, and PB suspensions, lysine production further increased by 12.3, 21.3, and 12.4% more than the cases of adding SL only during 12 h incubation, respectively. SL and B(6) were demonstrated to enhance the production of lysine from DAP-SI by mixed rumen protozoa, mixed rumen bacteria and their mixture in this study.     

Patterns of Methionine and Lysine Biosynthesis in the Trypanosomatidae During Growth*

SYNOPSIS. Nine Crithidia spp., 2 Blastocrithidia spp., 3 Leptomonas spp. and 2 Trypanosoma spp. were tested for ability to synthesize methionine and lysine during growth. A prerequisite for methionine biosynthesis is an inordinately high level of folic acid (0.1 mg/100 ml) in the medium. Crithidia factor-type unconjugated pteridines cannot spare this requirement. Since the methionine-synthesis factor is still present after acid hydrolysis which destroys folic acid, the factor is either a breakdown product of folic acid or an impurity in the commercial product. All save C. fasciculata var. noelleri and C. from Syrphid could synthesize methionine from homocysteine thiolactone. None of the organisms synthesized lysine from α-aminoadipic acid (AAA), thus ruling out the existence of the AAA pathway for lysine synthesis in the Trypanosomatidae. Nine of the organisms synthesized lysine from a mixture of LL- and meso-α,e-diaminopimelic acid. Since both LL- and meso-DAP are intermediates in the biosynthesis of lysine by the DAP pathway (LL-DAP→meso-DAP→lysine) and since decarboxylation of either LL- or meso-DAP could result in formation of lysine, pure meso-DAP was tested and found active. Thus at least the terminal portion of the DAP pathway for lysine synthesis exists in these true animal cells. Statements about absence of ability to synthesize lysine in animal cells and consequent evolutionary interpretations will therefore require revision.     

Crystal Structure of Diaminopimelate Epimerase from Arabidopsis thaliana, an Amino Acid Racemase Critical for l-Lysine Biosynthesis

Diaminopimelate (DAP) epimerase is a key enzyme for the biosynthesis of lysine in plants. Lysine is an essential dietary nutrient for mammals. In both plants and bacteria, DAP epimerase catalyzes the interconversion of ll-DAP and dl(meso)-DAP. The absence of a mammalian homolog makes DAP epimerase a promising target for the design of novel herbicides and antibacterials. This enzyme requires no cofactors and it functions through an unusual mechanism involving two cysteine residues acting in concert and alternating as a base (thiolate) and as an acid (thiol). The present study reports the crystal structures of two enzyme-inhibitor complexes of DAP epimerase from Arabidopsis thaliana with different isomers of the irreversible inhibitor and substrate mimic, 2-(4-amino-4-carboxybutyl)-aziridine-2-carboxylate, at 1.95 and 2.3 Å resolution. These structures provide the first atomic details of a plant amino acid racemase. Structural analysis reveals that ligand binding to a cleft between the two domains of the enzyme is accompanied by domain closure with two strictly conserved cysteine residues, Cys99 and Cys254, optimally positioned to perform acid/base catalysis via a carbanion stabilization mechanism on the stereogenic α-carbon atom of the amino acid. Stereochemical control in catalysis is achieved by means of a highly symmetric catalytic site that can accommodate both the l and d stereogenic centers of DAP at the proximal site, whereas specific interactions at the distal site require only the l configuration. Structural comparisons of the plant enzyme with its bacterial counterpart from Haemophilus influenzae reveal significant conservation of amino acid residues around the active site that extends to their three-dimensional structures and catalytic mechanism.     

Isolation of bioactive actinomycetes from marine sediments using rifampicin

Summary Bioactive actinomycetes were isolated from marine sediments using rifampicin. Plating the sediments on starch-casein agar, supplemented with rifampicin, eliminated the occurrence of contaminating microorganisms. Total counts, however, were reduced in the presence of rifampicin. Most of the isolates contained ll-2,6-diaminopimelic acid (DAP), whereas 37% contained meso-DAP. The use of increasing concentrations of rifampicin tended to yield a higher proportion of strains with cell extracts positive for meso-DAP. Streptomyces and Micromonospora represented the major genera identified. Antimicrobial activity was exhibited by 46% of the isolates, primarily against Gram-positive bacteria. Inhibition of Gram-negative bacteria was minimal, but antimycotic activity was displayed by 28% of the actinomycetes. Most of the latter activity was attributable to polyenes, particularly hexanenes. The results obtained indicate that rifampicin, added to starch-casein agar, is effective for the isolation of bioactive actinomycetes from marine sediments.     

Occurrence of diaminopimelic epimerase in maize

Extracts of maize leaves catalyzed the interconversion of meso-diaminopimelic acid its L-isomer. Three observations support the existence of this epimerase activity: (i) detection of the reversible interconversion of L-diaminopimelic acid and meso-diaminopimelic acid by paper chromatography after incubation of either isomer with extract; (ii) formation of [¹⁴C]CO₂ from L-[¹⁴C]diaminopimelic acid in an incubation mix containing meso-diaminopimelic acid decarboxylase; and (iii) inhibition of [¹⁴C]CO₂ evolution from L-diaminopimelic acid by unlabeled meso-diaminopimelic acid. The demonstration of the diaminopimelic acid epimerase lends support to the occurrence in plants of the complete diaminopimelic acid pathway for biosynthesis of lysine as it occurs in Escherichia coli and most bacteria.     

In vitro production of lysine from 2,2'-diaminopimelic acid by rumen protozoa.

Rumen protozoa can produce lysine from free 2,2'-diaminopimelic acid (DAP). However, the quantitative importance of this transformation has been disputed; lysine contents of protozoal incubation supernatants reported by Onodera & Kandatsu and Masson & Ling show a 26-fold difference. The in vitro experimental methods of both groups were compared to determine the causes of this difference. Lysine production was proportional to DAP concentration. Results with rumen protozoa from sheep or goats were similar. The incubation medium and deproteinizing procedure of the Welsh group gave a two-fold increase in lysine production compared with Japanese protocols. Omissions of rice starch from protozoal incubations slightly increased lysine production, whereas omissions of antibacterial agents resulted in varying, yet relatively small changes. The greatest cause of the difference was the number of rumen protozoa incubated. When this factor was taken into account, the difference in the maximum rates of lysine production between the Welsh and Japanese groups was only three-fold, namely 4.5 versus 15.0 nmol lysine/10(5) protozoa/h. Adding other amino acids to the incubations suggested that DAP uptake by rumen protozoa may occur via transport system ASC. The importance of DAP metabolism by protozoa as a source of lysine for ruminant host animals is discussed.     

In Vitro Production of Lysine from 2,2'-Diaminopimelic Acid by Rumen Protozoa

Rumen protozoa can produce lysine from free 2,2'-diaminopimelic acid (DAP). However, the quantitative importance of this transformation has been disputed; lysine contents of protozoal incubation supernatants reported by Onodera & Kandatsu [12] and Masson & Ling [9] show a 26-fold difference. The in vitro experimental methods of both groups were compared to determine the causes of this difference. Lysine production was proportional to DAP concentration. Results with rumen protozoa from sheep or goats were similar. The incubation medium and deproteinizing procedure of the Welsh group gave a two-fold increase in lysine production compared with Japanese protocols. Omissions of rice starch from protozoal incubations slightly increased lysine production, whereas omissions of antibacterial agents resulted in varying, yet relatively small changes. The greatest cause of the difference was the number of rumen protozoa incubated. When this factor was taken into account, the difference in the maximum rates of lysine production between the Welsh and Japanese groups was only three-fold, namely 4.5 versus 15.0 nmol lysine/10⁵ protozoa/h. Adding other amino acids to the incubations suggested that DAP uptake by rumen protozoa may occur via transport system ASC. The importance of DAP metabolism by protozoa as a source of lysine for ruminant host animals is discussed.     

Catabolism of Lysine by Mixed Rumen Bacteria

Metabolites arising from the catabolism of lysine by the mixed rumen bacteria were chromatographically examined by using radioactive lysine. After 6 hr incubation, 241 nmole/ ml of lysine was decomposed to give ether-soluble substances and CO₂ by the bacteria and 90 nmole/ml of lysine was incorporated unchanged into the bacteria. δ-Aminovalerate, cadaverine or pipecolate did not seem to be produced from lysine even after incubation of the bacteria with addition of those three amino compounds to trap besides lysine and radioactive lysine. Most of the ether-soluble substances produced from radioactive lysine was volatile fatty acids (VFAs). Fractionation of VFAs revealed that the peaks of butyric and acetic acids coincided with the strong radioactive peaks. Small amounts of radioactivities were detected in propionic acid peak and a peak assumed to be caproic acid. The rumen bacteria appeared to decompose much larger amounts of lysine than the rumen ciliate protozoa did.     

Cloning and Sequencing of the meso-Diaminopimelate-d-dehydrogenase (ddh) Gene of Corynebacterium glutamicum

The ddh gene of Corynebacterium glutamicum encoding raesodiaminopimelate meso-diaminopimelate (meso-DAP)-d-dehydrogenase (DDH) involved in the lysine biosynthesis was cloned in a DAP auxotroph (dapD4) of Escherichia coli by complementation of the DAP auxotroph. Deletion analysis revealed that a ~1.7-kb XhoI-KpnI fragment contained the ddh structure gene. The specific activity of DDH was increased fourteen-fold when C. glutamicum was transformed with a recombinant plasmid harbouring the cloned ddh gene. Furthermore, the ddh gene has been sequenced [S. Ishino et al., Nucleic Acids Res., 15, 3917 (1987)] and some properties of the ddh gene are discussed.     

Formation of Lysine from α,ε-Diaminopimelic Acid and Negligible Synthesis of Lysine from Some Other Precursors by Rumen Ciliate Protozoa

The possibility of lysine formation from α,ε-diaminopimelate (DAP), acetate, aspartate or α-aminoadipate (AAA) in rumen ciliates was examined. DAP-1,7-¹⁴C added to the medium was decarboxylated and converted to radioactive lysine in great amounts and radioactive pipecolate in small amounts by rumen ciliates. Difference of the ability to form lysine from DAP between genus Entodinium and Diplodinium was not observed. With sodium acetate-U-¹⁴C, amino acids fraction of the supernatant fluid of the incubation medium and ciliates contained only 0.56 and 0.59% of the total radioactivity, respectively. In the case of l-aspartate-U-¹⁴C, 95.1% of the radioactivity of the supernatant fluid desalted and 62.2% of the radioactivity incorporated into ciliates (1.5% of the total radioactivity) remained as aspartate. Autoradiograms revealed the negligible spots of lysine in ciliates in both cases. AAA-6-¹⁴C remained almost unchanged, even after incubation with rumen ciliates.     

Biosynthesis of lysine in plants: the putative role of meso-diaminopimelate dehydrogenase

Extracts from Chlamydomonas, corn, soybean and tobacco were tested for enzymes of the lysine biosynthetic pathway. Dihydrodipicolinic acid (DHD) synthase, DHD reductase, diaminopimelate (DAP) epimerase and DAP decarboxylase were present in all. However, in contrast to the report of Wenko et al., meso-DAP dehydrogenase could not be detected in extracts prepared from soybean. Moreover, it was not found in Chlamydomonas, corn and tobacco as well. In order to set an upper limit to the amount of meso-DAP dehydrogenase that might be present, reconstruction experiments were performed with soybean and corn extracts in which the conversion of dihydrodipicolinate to lysine was made dependent on the addition of limited amounts of the meso-DAP dehydrogenase purified from Bacillus sphaericus. The presence of DAP epimerase and the absence of meso-DAP dehydrogenase indicates that the meso-DAP dehydrogenase abbreviated pathway for lysine synthesis is not operative in plants.     

Biosynthesis of Threonine from Homoserine by Mixed Rumen Microorganisms: An In Vitro Study

The biosynthesis of threonine (Thr) by using the main biosynthetic pathway involving homoserine (Hser) was quantitatively investigated by mixed rumen bacteria (B), protozoa (P), and their mixture (BP) in an in vitro system. Rumen contents were collected from fistulated goats to prepare the microbial suspensions and were incubated anaerobically at 39°C for 12 h with or without Hser (2 mm) as a substrate. Thr and other related compounds produced in both the supernatants and hydrolysates of the incubation were analyzed by HPLC. During a 12-h incubation period, 84.2%, 58.1%, and 92.0% of Hser disappeared in B, P, and BP suspensions, respectively. Rumen bacteria and the mixture of rumen bacteria and protozoa were demonstrated for the first time to produce Thr from Hser, and the production of Thr from Hser in BP (371.9 and 297.2 μmol/g MN) (MN, microbial nitrogen) was about 13.0% and 9.1% higher than that in B alone (329.2 and 272.5 μmol/g MN) during 6- and 12-h incubations, respectively. On the other hand, mixed rumen protozoa were unable to synthesize Thr from Hser. Other metabolites produced from Hser were found to be glycine (Gly) and 2-aminobutyric acid (2AB) in B and BP. In P, Gly and 2AB were not found. The results mentioned above indicated the abilities of rumen bacteria and the mixture of rumen bacteria and protozoa to synthesize Thr de novo from Hser and appeared as first-time report. Received: 24 May 2000/Accepted: 4 August 2000     

Production of tyrosine and other aromatic compounds from phenylalanine by rumen microorganisms

Summary Rumen contents from three fistulated Japanese native goats fed Lucerne hay cubes (Medicago sativa) and concentrate mixture were collected to prepare the suspensions of mixed rumen bacteria (B), mixed protozoa (P) and a combination of the two (BP). Microbial suspensions were anaerobically incubated at 39°C for 12h with or without 1 MM ofl-phenylalanine (Phe). Phe, tyrosine (Tyr) and other related compounds in both supernatant and microbial hydrolysates of the incubations were analyzed by HPLC. Tyr can be produced from Phe not only by rumen bacteria but also by rumen protozoa. The production of Tyr during 12h incubation in B (183.6 mol/g MN) was 4.3 times higher than that in P. One of the intermediate products between Phe and Tyr seems to bep-hydroxyphenylacetic acid. The rate of the net degradation of Phe incubation in B (76.O mol/g MN/h) was 2.4 times higher than in P. In the case of all rumen microorganisms, degraded Phe was mainly (>53%) converted into phenylacetic acid. The production of benzoic acid was higher in P than in B suspensions. Small amount of phenylpyruvic acid was produced from Phe by both rumen bacteria and protozoa, but phenylpropionic acid and phenyllactic acid were produced only by rumen bacteria.     

RppH-dependent pyrophosphohydrolysis of mRNAs is regulated by direct interaction with DapF in Escherichia coli

Similar to decapping of eukaryotic mRNAs, the RppH-catalyzed conversion of 5′-terminal triphosphate to monophosphate has recently been identified as the rate-limiting step for the degradation of a subset of mRNAs in Escherichia coli. However, the regulation of RppH pyrophosphohydrolase activity is not well understood. Because the overexpression of RppH alone does not affect the decay rate of most target mRNAs, the existence of a mechanism regulating its activity has been suggested. In this study, we identified DapF, a diaminopimelate (DAP) epimerase catalyzing the stereoinversion of L,L-DAP to meso-DAP, as a regulator of RppH. DapF showed a high affinity interaction with RppH and increased its RNA pyrophosphohydrolase activity. The simultaneous overexpression of both DapF and RppH increased the decay rates of RppH target RNAs by about a factor of two. Together, our data suggest that the cellular level of DapF is a critical factor regulating the RppH-catalyzed pyrophosphate removal and the subsequent degradation of target mRNAs.     

Effects of protozoa on Methane production in rumen and hindgut of calves around time of weaning

Effects of the presence or absence of ciliate protozoa on methanogenesis in the rumen and hindgut were investigated in young calves during a 7-week period. Ten Holstein calves, aged 7 days, were divided in two groups (n = 5) and fed an increasing amount of a commercial milk replacer and small amounts of a calves starter. One group was inoculated with ciliate fauna on two occasions, week 5 and 6, while the second remained ciliate-free. The absence of protozoa in the rumen decreased rumen empty weight ( ? 23%, P < 0.01), and rumen pool size of N ( ? 36%, P < 0.01) and crude fat ( ? 37%, P < 0.05). Rumen bacteria of non-faunated calves contained a higher proportion of total amino acid-N per 16 g N ( + 3%, P < 0.01) and D-alanine-N per 16 g N ( + 13%, P < 0.05) compared to faunated calves. Further results contain a reference for a higher bacterial mass in the ciliate-free rumen with an increased number of bacteria adherent to rumen mucosa. The CH₄ production in the rumen increased exponentially with the increase in protozoa population size (R² = 0.68). In presence of 46 · 10⁴ protozoa per ml rumen fluid, the in vitro CH₄ production of rumen fluid per mol total VFA was about 34% higher in faunated than in non-faunated calves (P < 0.001). Hydrogen (2H) recovery of rumen fermentation was positively correlated (R² = 0.55) to the CH₄ production rate. Methanogens were attached on rumen mucosa. Methanogenesis, induced by rumen mucosa attached bacteria, was stimulated by ruminal protozoa. In the absence of protozoa in the rumen, the acetate - propionate ratio and butyrate proportion of VFA were reduced. In vivo in the absence of protozoa not only the whole animal CH₄ production ( ? 30%, P < 0.05) but also the digestibility of carbohydrates ( ? 4%, P < 0.05) was reduced. Thereby no difference was observed in the intake of ME per kg DM between the groups. In conclusion, the methanogenesis in the rumen, but not in hindgut, is associated with the development of the ruminal protozoa population. The level of methanogenesis (mol/mol VFA) in the hindgut amounts to 20% of the ruminal methanogenesis.     

Thin layer chromatographical detection of tyrosine produced from <Emphasis Type="SmallCaps">l</Emphasis>-[U-<Superscript>14</Superscript>C]phenylalanine by ruminal microbes

Summary.  Thin layer chromatographical detection of tyrosine (Tyr) synthesized from l-[U-¹⁴C]phenylalanine (Phe) (1 mM) by rumen bacteria (B) and protozoa (P) collected from fistulated Japanese Goat was carried out. About 16 and 12% of the added Phe was converted to Tyr by B and P, respectively. Large amount of radioactivity in ether fractions indicated an abundant production of aromatic acids from Phe. Small amount of radioactivity found in CO₂ fractions implied an occurrence of considerable decarboxylation reaction(s) by rumen bacteria and protozoa. Received July 18, 2001 Accepted December 3, 2001     

The importance of methanogens associated with ciliate protozoa in ruminal methane production in vitro

The importance of methanogenic bacteria associated with ciliate protozoa was estimated either by removing protozoa from whole rumen fluid (using defaunated rumen fluid to correct for the effects of centrifugation on bacteria) or by isolating the protozoa. Rumen fluid was withdrawn from sheep inoculated with either Polyplastron multivesiculatum , a co-culture of Isotricha prostoma plus Entodinium spp. or a mixed type B fauna of Entodinium, Eudiplodinium and Epidinium spp. Methanogenesis was highest in rumen fluid containing a mixed protozoal population of the following genera: Entodinium, Eudiplodinium and Epidinium , was lower in defaunated rumen fluid and lowest in rumen fluid containing either I. prostoma plus Entodinium or P. multivesiculatum . Methanogenic bacteria associated with rumen ciliates were apparently responsible for between 9 and 25% of methanogenesis in rumen fluid.