Amino acid biosynthesis

The rumen microbes depend to a large extent on de novo synthesis of amino acids from ammonia and carbon precursors derived from products of carbohydrate fermentation (Sauer et al., 1975). Amino acid families are based on the source of carbon used for their synthesis (Wallace et al., 1997): the glutamate family - glutamate, glutamine, proline, arginine; the serine family - serine, glycine, cysteine; the aspartate family - aspartate, lysine, methionine, threonine, isoleucine; the pyruvate family - alanine, isoleucine, leucine, valine; the aromatic family - phenylalanine, tyrosine, tryptophan and histidine. Although the pathways of amino acid synthesis in ruminal bacteria have not been studied to the same extent as in enteric bacteria, evidence is available for a number of these pathways and radioactive tracer studies on amino acid biosynthesis by mixed ruminal bacteria provided amino acids with labelling patterns consistent with the pathways described by Umbarger (1978) for the synthesis of amino acids by bacteria and fungi (Sauer et al., 1975; Wallace et al., 1997). As described above, glutamate plays a central role in the nitrogen metabolism of organisms, and thus the generation of a-ketoglutarate is of great importance to nitrogen metabolism in ruminal bacteria (Wallace et al., 1997). Since the rumen is an anaerobic environment, ruminal microbes contain an incomplete Krebs cycle, in which a-ketoglutarate is not generated as an intermediate of energy metabolism as it is in aerobic organisms (Patterson, 1992). Milligan (1970) demonstrated the labelling of glutamate in the C-l, C-2 and C-5 positions when ruminal contents were incubated with NaH14C03. This labelling pattern suggested that a-ketoglutarate is formed by both reductive carboxylation of succinic acid for the reverse Krebs cycle and condensation of oxaloacetic acid and acetyl CoA to form

Table 15.2. Effect of peptide concentration on the GDH activity of Prevotella bryantii, Selenomonas ruminantium and Streptococcus bovisP. (Atasoglu and Wallace, unpublished data.)

P. bryantii S. ruminantium S. bovis

Table 15.2. Effect of peptide concentration on the GDH activity of Prevotella bryantii, Selenomonas ruminantium and Streptococcus bovisP. (Atasoglu and Wallace, unpublished data.)

P. bryantii S. ruminantium S. bovis

Medium

NADP

NAD

NADP

NAD

NADP

NAD

Basal

3.53 ± 0.42

0.11 ±0.02

3.65 ±0.91

NDC

3.16 ±0.58

ND

Basal + 1 g r1

Tryd

2.72 ± 0.39

0.10 ±0.02

2.74 ± 0.68

ND

2.55 ±0.19

ND

Basal + 5 g I 1

Try

1.80 ± 0.37

0.06 ±0.01

1.06 ±0.06

ND

1.19 ± 0.10

ND

Basal + 10 g I

1 Try

1.28 ± 0.03

0.04 ±0.01

1.43 ±0.07

ND

0.92 ±0.18

ND

Basal + 30 g I

1 Try

1.20 ± 0.33

0.03 ±0.01

1.04 ±0.21

ND

0.84 ± 0.20

ND

Basal + 10 g I

1 Trye

0.87 ±0.17

0.02 ±0.01

1.09 ±0.19

ND

0.94 ± 0.03

ND

Medium M2

1.20 ± 0.22

0.03 ± 0.00

2.41 ±0.32

ND

0.36 ± 0.01

ND

aResults are the means of triplicate cultures.

bNAOP and NAD-linked specific activity is defined as |j.mol of NAD(P)H oxidized min 1 mg 1 of protein. CND, not detectable. dTry, trypticase.

eBasal medium contained 10 g I ' trypticase without added ammonia.

citrate and subsequent forward Krebs cycle activities. In addition, the continuous culture study with mixed ruminai microorganisms by Sauer et al. (1975) provided evidence for the synthesis of a-ketoglutarate by forward and reverse Krebs cycle reactions. The presence of reductive carboxylation of succinate to form a-ketoglutarate was demonstrated by Allison and Robinson (1970) in P. ruminicola as well as in Veillonella, Seleriomorias and Bacteroides spp. (Allison et al., 1979). Likewise, when cells of F. succinogenes were incubated with [l-13C]glucose, 13C-labelled Asp, Glu, Ala and Val were detected by Matheron et al. (1999), who concluded that the labelling of amino acids was consistent with the proposed amino acid synthesis pathway and with the reversal of the succinate synthesis pathway.

Biosynthesis of alanine results from the amination of pyruvate and can be catalysed by alanine dehydrogenase and glutamate-pyru-vate transaminase using glutamate as the nitrogen donor. Pyruvate is generated in the energy metabolism of the majority of ruminai bacteria and can be produced by the reductive carboxylation of acetate (Allison, 1969). Serine is produced from phosphoglyceric acid, which is a glycolytic intermediate, by conversion of this compound into phosphohy-droxypyruvate and then phosphoserine and serine (Sauer et al., 1975). Aspartate is formed from oxaloacetate and ammonia by aspartate dehydrogenase or by the glutamate-oxaloacetate transaminase reaction using glutamate as the nitrogen donor (Wallace et al., 1997; Morrison and Mackie, 1997).

Experiments in which bacterial amino acids were labelled with 15N revealed that glutamate and alanine, together in most experiments with aspartate, were the most rapidly labelled amino acids with 15N, confirming the importance of these amino acids as the initial recipients of amino groups for subsequent transfer to other amino acids in the ruminai microorganisms (Salter et al., 1979; Atasoglu et al., 1999). Glutamate was found to be the most abundant amino acid in the free amino acid pool into which ammonia would be assimilated (Wallace, 1979; Blake et al., 1983). High enrichment of glutamate with 15N was also observed in ruminai microorganisms, which is consistent with glutamate dehydrogenase being the main ammonia-assimilating enzyme in ruminai bacteria, as described above. However, despite the low activity of alanine dehydrogenase and gluta-mate-pyruvate aminotransferase (Wallace, 1979), alanine was surprisingly observed to be prominent in these pools and often exceeded glutamate, particularly when ammonia concentrations were high. [15N]Ammonium chloride enriched alanine more than glutamate or other amino acids in the microbial pool after only 2 min (Blake et al., 1983), implying that alanine dehydrogenase is extremely active under the conditions of high ammonia concentrations, consistent with the findings of Wallace (1979). Other investigators, using 15N-labelled urea or ammonium chloride also found alanine to be one of the most labelled amino acids (Shimbayashi et al., 1975; Blake et al., 1983; Atasoglu et al., 1999). Blake et al. (1983) postulated that alanine may be the primary product and high concentration of alanine may be important to the ruminai bacteria as a short term storage mechanism for ammonia, as a control mechanism within the bacterium to prevent excess levels of ammonia accumulation, and a route of removing excess pyruvate from the bacterium when available energy is in excess and bacterial metabolism is rapid.

Phenylacetate, hydroxyphenylacetate, and indoleacetate are substrates for reductive carboxylation and can be used for the biosynthesis of phenylalanine, tyrosine and tryptophan, respectively (Allison, 1969). The reductive carboxylation pathways present in some ruminai bacteria appear to be in addition to the biosynthesis of these aromatic amino acids from a common precursor, cho-rismate (Morrison and Mackie, 1997). The biosynthesis of chorismate requires phospho-enolpyruvate and erythrose-4-phosphate as substrates and proceeds via the shikimate pathway. The presence of both pathways for the biosynthesis of aromatic amino acids was also demonstrated by Sauer et al. (1975). Interconversion of aromatic amino acids also occurs in the rumen, tyrosine being formed from phenylalanine (Khan et al., 1999)

Ruminai bacteria are known to synthesize branched-chain amino acids from branched-chain fatty acids (Allison, 1969). Isovalerate, 2-

methylbutyrate and isobutyrate are reductively carboxylated, then aminated to produce leucine, isoleucine and valine, respectively. One or more of these branched-chain fatty acids is required for the synthesis of branched-chain amino acids by the predominant cellulolytlc ruminal bacteria, Fibrobacter succinogenes, Ruminococcus albus and R. flavefacieris (Bryant and Robinson, 1962; Allison et al., 1962). Prevotella ruminicola and Megasphaera els-denii, which produce branched-chain fatty acids, can also utilize these for the synthesis of amino acids (Allison, 1969). In rumen bacteria, the availability of branched-chain fatty acids has also been demonstrated to modulate the flux of glucose carbon into amino acids. Allison et al. (1984) found that growing cultures of R ruminicola utilized carbon from [14C]glucose for the synthesis of leucine and other cellular amino acids when the growth medium was not supplied with isovalerate. When unlabelled iso-valerate was available, however, utilization of [14C]glucose for leucine synthesis was markedly reduced. Similarly, provision of phenylacetate and 2-methylbutyrate reduced the utilization of glucose carbon for phenylalanine and isoleucine synthesis, respectively (Allison et al., 1984). The authors concluded that this organism has the ability to regulate alternative pathways for the synthesis of certain amino acids, and will utilize preformed intermediates of these amino acids in preference to de novo synthesis. Furthermore, because these intermediates are often present in the rumen fluid, pathways that involve reductive carboxylations are likely to be predominant for the synthesis of these amino acids in the rumen (Allison et al., 1984; Wallace et al., 1997). 15N enrichment in valine, leucine and isoleucine was lower than that of most of the other amino acids in mixed ruminal microorganisms, when peptides or amino acids were present in the growth medium (Atasoglu et al., 1999), a finding consistent with the conclusion of Allison et al. (1984) for P. ruminicola.

The glutamate family of amino acids includes proline and arginine. The biosynthesis of these amino acids is likely to be similar to the pathways described for the enteric bacteria (Morrison and Mackie, 1997). The biosynthesis of proline and arginine requires that the 7-carboxyl group of glutamate be acti vated, then reduced, to yield glutamic-7-semi-aldehyde (Morrison and Mackie, 1997). Glutamic^Y-semialdehyde gives rise to pyrro-line-5-carboxylate, which is reduced to proline. The semialdehyde also gives rise to ornithine, which is converted into arginine.

Other examples of amino acids being formed from C-skeleton precursors in the rumen include histidine from imidazole compounds (Wadud et al., 2001), threonine from homoserine (Or-Rashid et al., 2001), lysine from diaminopimelic acid by protozoa (Onodera and Kandatsu, 1973; Onodera 1986), and tryptophan from indole pyruvic acid (Okuuchi et al., 1993).

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