Nitrogen assimilation

The possible nitrogen sources range from molecular nitrogen and inorganic compounds (such as nitrate) to amino acids (such as tryptophan, histidine, proline,

arginine, ornithine, and serine). Enzymatically catalysed transformations of these different compounds yield ammonia. The key substances mediating the assimilation

of ammonia into the different nitrogenous cellular compounds are glutamate and its amide, glutamine (Tyler, 1978; Magasanik, 1982). Some of the amino acids capable

of serving as sources of nitrogen, such as proline, histidine, and arginine, can be

directly metabolised to glutamate (Privai and Magasanik, 1971; Friedrich and Magasanik, 1978).

INTRODUCTION Nitrogen in Microbiai Process

Glutamate serves as the amino donor in transamination reactions with appropriate keto

acids, catalysed by several aminotransferases, leading to the biosynthesis of the

different amino acids (Deshpande and Kane, 1980). Glutamine, which is the amino

acid donor in the biosynthesis of purines, histidine, and tryptophan, is itself the

product of y-glutamyl transfer to ammonia catalysed by the enzyme glutamine

synthetase (GS) (Meister, 1956).

In view of the key role of glutamate and glutamine in nitrogen metabolism, much

attention has been devoted to their biosyntheses, and its associated enzymology, and to the physiological and genetic factors controlling the rates of these reactions.

However in what follows, the role of glutamate and glutamine has focused on the assimilation of ammonia and ammonium ions.

Ammonium ions are assimilated by reductive amination of 2-ketoglutarate with the formation of glutamate catalysed by a glutamate dehydrogenase (GDH) in the presence of a reduced coenzyme (NADPH). GDH is widely known as the key enzyme responsible for the assimilation of ammonia into cellular nitrogenous components (Halpem and Umbarger, 1960). GDH catalyzes the Reaction 1.10.

2-ketoglutarate + N H / + NADPH <--- ► glutamate + NADP^ (1.10)

A second reaction, where ammonium ions are assimilated, is the amido synthesis of glutamine by glutamine synthetase (GS) in the presence of glutamate and ATP. The

Reaction 1.11 is catalysed by GS.

glutamate+ N H / +ATP ■■ glutamine + H^ + ADP + Pj (1.11)

These two enzymes are usually present in all microorganisms and, although the enzymes present some differences in the genes, polypeptides, oligomers, and post-

translational modification, each one of these enzymes catalyses the same type of action (Meister, 1980).

INTRODUCTION Nitrogen in Microbial Process

In addition, another reaction, reported as an alternative route to glutamate

biosynthesis, cannot be ignored, although it was not found directly related to

ammonium ions assimilation. This reaction was very similar to the one catalysed by GDH. It is catalysed by aminotransferase oxidoreductase (GOGAT, glutamate

synthase) in that glutamine and not ammonium ions served as amino donor as shown

in the Reaction 1.12 (Meers et al, 1970).

2-ketoglutarate + glutamine + NADPH —> 2 glutamate + NADP"^ (1.12)

Comparison of the net sum of the combined reactions of GS and GOGAT to that of

GDH reveals a very interesting and highly significant point. In both systems, one molecule of 2 -ketoglutarate is converted to glutamate, with one molecule of

ammonium ion being assimilated in the process and one molecule of NADPH being

oxidised. However, in GS-GOGAT reaction cycle, as shown below (Reaction 1.13) there is an additional expenditure of one energy-rich phosphate bond of ATP necessary to form the amide bond of glutamine.

2-ketoglutarate + N H / + NADPH + ATP —> glutamate + NADP^ + H^ + ADP + Pj

(1.13) Due to the concomitant hydrolysis of ATP, the combined reactions of glutamate

synthase-glutamine synthetase have a favourable equilibrium allowing the utilisation of ammonia present in the medium in the micromolar concentration range. The more

economical glutamate dehydrogenase reaction has an unfavourable equilibrium and is

therefore physiologically significant only with ammonia concentrations in the medium in the millimolar range (Magasanik, 1982; Brenchley and Magasanik, 1974; and

Brenchley et a l, 1973). Mutants lacking the glutamate synthase (GOGAT) activity cannot grow in minimal media with concentrations of ammonia below 0.1 mM, but

grow well at higher ammonia concentrations. Mutants lacking the glutamate

dehydrogenase (GDH) activity are not impaired in their ability to grow in ammonia- minimal media. And the mutants lacking both GOGAT and GDH are glutamate

auxotrophs (Berberich, 1972; Berberich, 1973).

INTRODUCTION Nitrogen in Microbial Process

In bacterial cultures, when ammonia concentration is limiting, the function of

glutamine, which serves as the amino donor for the biosynthesis of glutamate by

GOGAT, becomes very important even if GDH is present in great amounts. In

ammonia-starved cultures, the bulk of nitrogen flux is handled by GS, with close to

90% of the assimilated nitrogen passing through glutamate via the GOGAT reaction

(Wohlhueter e/a/., 1973).

1.6.1.1

Ammonium ion assimilation in Streptomycetes

As mentioned above, ammonium assimilation in bacteria is known to be mediated

mainly by two mechanisms: (1) the reductive amination of 2 -ketoglutarate to yield

glutamate, catalysed by glutamate dehydrogenase (GDH), and (2) formation of glutamine from glutamate and ammonium, catalysed by glutamine synthetase (GS),

followed by the transfer of the amide group to 2 -ketoglutarate, catalysed by glutamate

synthase (GOGAT), which results in the net synthesis of one glutamate molecule. Some bacteria seem to possess only the second mechanism. The participation of other amino acid dehydrogenases, like alanine dehydrogenase (ADH), in ammonium

assimilation is less well documented, but because of their high for ammonium, they might be significant only when the environmental concentration o f this ion is high (Brana and Demain, 1988).

Several reports have indicated the presence of the above enzymes in different species

of streptomycetes, but only a few cases is there evidence of the pathways that are actually functional. This is of interest from the point of view of nitrogen repression of

antibiotic synthesis, since the interference by ammonium or other nitrogen sources

may be related to the enzymes or the immediate products of nitrogen assimilation. In S. clavuligerus, significant levels of GS, GOGAT, and ADH were detected in crude extracts of this organism growing in different media (Brana et al, 1986; Aharanowitz, 1979; Aharonowitz and Friedrich, 1980). GS activity varied markedly depending on

the nitrogen source, although repressed levels were always found in the presence of

ammonium.

INTRODUCTION Nitrogen in Microbiai Process

GOGAT activities were rather constant, independent of the nitrogen source, whereas

ADH was induced when high ammonium or alanine concentrations were present in the

culture medium. GDH and alanine-2-keto-glutarate aminotransferase were not found

under any growth conditions. After mutagenesis, the data indicated that the GS-

GOGAT pathway was the only means of ammonium assimilation in S. clavuligerus, ruling out a direct role of ADH in this process. There are scattered reports on the presence of the enzymes of ammonium assimilation in streptomycetes. Given the high

number of known strains and their relative diversity, it is quite likely (as with bacteria

in general) that some of them will have both GS-GOGAT and GDH pathways, while

others use only the first one.

As it plays the central role in nitrogen control, the specific characteristics of GS firom streptomycetes are described here. GS activities are depressed by ammonium ions in many Steptomyces sp., although a slight increase in activity with very high ammonium concentration was observed in S. clavuligerus (Brana et al, 1986). Various amino acids also produced some increases in activity in several Streptomyces sp. growing with ammonium. However, the complexity of the control mechanisms that affect GS in other bacteria signals caution in interpreting these results. A series of mechanisms including repression-derepression, reversible inactivation, availability of divalent

cations, and feedback inhibition can all influence GS activity (Shapiro and Stadtman, 1970). In addition, the enzyme from streptomycetes seems to respond to the presence

of divalent cations and pH changes in a way different from that o f GS from enteric bacteria, in which it serves as an indicator of nitrogen regulation. As a consequence,

the actual amount of GS cannot be measured in streptomycetes following the usual procedures (Bender et al, 1977), but if the reported activities reflect the real levels of enzyme in the cells, the effect of ammonium would be mainly a repressive one.

INTRODUCTION Nitrogen in Microbial Process

1.6.1.2

Nitrogen assimilation and antibiotic production

In the absence of detailed studies on nitrogen metabolite repression in actinomycetes,

there have been repeated attempts to relate nitrogen control of primary metabolism

and antibiotic production. Circumstantial evidence of a correlation between

ammonium interference of antibiotic synthesis and GS activity (Wax et al, 1982) in different micro-organisms has led to speculation about a possible regulatory network affecting nitrogen utilisation and secondary metabolism (Brana and Demain, 1988).

In S. clavuligerus, it was clearly seen that cephalosporin synthesis and the activity of p-lactam synthetases did not correlate with the levels of enzymes of ammonium ions

assimilation (Brana et al, 1986a). Although the elimination of GS and GOGAT activity prevented utilisation of ammonium as nitrogen source, ammonium ions still repressed p-lactam synthetases and antibiotic production in these mutants, whichever

of the three enzymes involved in the process was blocked. Similar conclusions that GS control differs from control of antibiotic formation were reached in the case of tylosin production by S. fradiae (Omura and Tanaka, 1985). Brana and Demain (1988) concluded that the data obtained concerning the enzymes of ammonium

assimilation and their immediate products indicated that the signal eliciting ammonium repression of cephalosporin biosynthesis is not transmitted through the pathways by which ammonium is assimilated in S. clavuligerus. The mechanisms of regulation might be more complex, and positive as well as negative signals may be involved.