OF PYRIMIDINE FERMENTATIONS BY Micrococcus aerogenes

Product

Substrate Product

Uracil Cytosine Thymine

Carbon dioxide 115 43 108

Hydrogen 55 12 39

Acetate 43 0 55

Lactate 67 24 72

Ammonia 169 131 99

Uracil — ^{78 29}

° The figures give moles of product per 100 moles of substrate fermented. Cell suspension experiments.

which has not yet been demonstrated. T h e reduced D P N formed in reac­

tion (42) could be used for the conversion of orotic acid to dihydro-orotic acid, the first step in the fermentation.

T h e final steps in the orotic acid fermentation appear to be an oxidation of pyruvate to acetate and carbon dioxide coupled with a reduction of oxalacetate via malate and fumarate to succinate. T h e postulated enzymic reactions have not been studied in detail.

Micrococcus aerogenes ferments uracil, cytosine, and thymine slowly and often incompletely.^{1 1 7} T h e main products are lactate, acetate, carbon diox­

ide, hydrogen, and ammonia (Table X I ) . Uracil is also formed during the decomposition of cytosine or thymine and appears to be an intermediate product. Nothing else is known about the chemistry of pyrimidine break­

down in Μ. aerogenes.

Clostridium uracilicum was isolated from soil by enrichment in a medium containing uracil and yeast extract.^{1 1 5} T h e organism requires biotin, several amino acids, and a carbohydrate for growth. Uracil is not required and does not stimulate growth. Uracil is decomposed only b y cells t h a t have grown in the presence of this compound. Other pyrimidines appear not to be attacked.

T h e products of uracil decomposition by adapted cell suspensions of C.

uracilicum are β-alanine, carbon dioxide, and a m m o n i a .^{1 5 0} Cell suspensions and cell-free extracts also convert dihydrouracil or β-ureidopropionic acid to the same products, and convert carbamylphosphate to orthophosphate, carbon dioxide, and ammonia. 0-Alanine is not decomposed. B o t h dihydro­

uracil and 0-ureidopropionic acid have been identified as intermediates in uracil breakdown by extracts. Furthermore, an enzyme which catalyzes the reversible reduction of uracil to dihydrouracil by D P N H has been demonstrated and partially purified. This reaction is analogous t o the

HN—CO HN—CO OC CH , ^{±DP NH}- OC CH, ± D P N +

H i - ! Η H N — C H²

Uracil Dihydrouracil

±HtO H²N COOH H²N C H²C H²C O O H + C 0² + N H³ < ^{+ H}' ° o i <^Η²

HN—(t)H

²

0-Alanine 0-Ureidopropionic acid

FIG. 8. Decomposition of uracil by Clostridium uracilicum.

reversible reduction of dihydro-orotic acid catalyzed by an enzyme from Z. oroticum. T h e accumulated evidence indicates t h a t the decomposition of uracil involves the reactions shown in Fig. 8 .

3. ALLANTOIN

Streptococcus allantoicus, a homofermentative lactic acid bacterium iso­

lated from an enrichment medium containing allantoin, ferments this com­

pound readily in a medium containing a little yeast extract.^{1 1 9} T h e prod­

ucts in moles per mole of allantoin decomposed are t h e following: ammonia, 2 . 2 6 ; urea, 0 . 6 2 ; oxamic acid, 0 . 4 5 ; carbon dioxide, 1 . 6 8 ; formate, 0 . 0 9 ; acetate, 0 . 1 5 ; glycolate, 0 . 1 4 ; and lactate, 0 . 0 1 . A small a m o u n t of glycine is also formed. Oxamic acid, the monoamide of oxalic acid, has not been observed elsewhere as a natural product.

A little information is available concerning t h e chemistry of allantoin fermentation. T h e first step appears to be t h e formation of allantoic acid

C O — N H COOH

NC O ^{+ H 2}° > H²N C O N H — C H — N H C O N H² (44)

/ H²N C O N H — C H — N H

Allantoin Allantoic acid

by a hydrolytic reaction. T h e latter compound is b o t h formed and decom­

posed a t a relatively rapid rate. T h e presence of 0 . 0 5 Μ sodium fluoride strongly inhibits t h e decomposition of allantoic acid and causes it to accu­

mulate during the breakdown of allantoin. F u r t h e r steps in the fermenta­

tion are not known. However, since S. allantoicus forms both ammonia and urea b u t does not contain the enzyme urease, it is apparent t h a t a t least one ureido group is decomposed b y a p a t h not involving urea. Tracer ex­

periments with N^{1 5}H³ have shown t h a t t h e nitrogen in oxamic acid is not

derived from ammonia; therefore oxamic acid m u s t be formed rather di­

rectly b y cleavage and oxidation of allantoic acid rather t h a n b y ammonol-ysis of a n oxalyl compound. Glyoxylate m a y be a n intermediate in t h e fermentation since it is readily decomposed anaerobically b y cell suspen­

sions. T h e products, other t h a n carbon dioxide, have not been identified.

T h e enzyme system involved in allantoin fermentation is adaptive. Cells grown on glucose are entirely inactive on allantoin.

4. NICOTINIC ACID

An unidentified Clostridium isolated b y t h e enrichment culture method was shown t o grow in a medium containing nicotinic acid, yeast extract, and peptone.^{1 1 6} Nicotinic acid is required for growth and t h e amount of growth is roughly proportional t o t h e concentration of nicotinic acid u p t o 0.2%.

T h e products of t h e anaerobic decomposition of nicotinic acid b y washed cell suspensions are ammonia, carbon dioxide, acetate, and propionate.

/

^\

*CH ³C — C O O H

I || + 4 H²0 N H³ + C 0² + C H 3 C O O H +

eC H *CH C H³C H²C O O H

\ /

^{( 4 5 )}

Nicotinic acid

T h e fermentation is rather accurately described b y equation (45). T h e only related compound fermented b y t h e organism is nicotinamide. Several other compounds, including pyridine, picolinic acid, isonicotinic acid, quinolinic acid, anthranilic acid, p-aminobenzoic acid, 3-hydroxyanthra-nilic acid, kynurenic acid, and iV-methylnicotinamide, are n o t attacked.

Some evidence concerning t h e chemical steps in t h e nicotinic acid fer­

mentation is available. Lyophilized bacteria contain an enzymic system t h a t couples t h e oxidation of nicotinic acid t o 6-hydroxynicotinic acid with the reduction of methylene blue.^{1 5 1} Tracer experiments have established t h a t nicotinic acid and 6-hydroxynicotinic acid are rapidly interconverted in t h e absence of a n external oxidizing agent according t o t h e reaction:

nicotinic acid + H²0 ^ 6-hydroxynicotinic acid + 2H (46) 6-Hydroxynicotinic acid is not definitely known t o be on t h e p a t h of nico­

tinic acid fermentation. However, it is possible t h a t 6-hydroxynicotinic acid is a n intermediate and is oxidized t o t h e corresponding ketone, 6-pyridone. T h e 6-pyridone theoretically could undergo a hydrolytic cleavage between t h e carbon in t h e 6 position and t h e nitrogen t o form a n acyclic compound from which t h e fermentation products could b e derived.

III. Fermentations of Pairs of Amino Acids (Stickland Reaction) Many Clostridia, growing on protein hydrolyzates or amino acid mix­

tures, appear t o obtain most of their energy b y a coupled oxidation-reduc­

tion between suitable amino acids, or amino acids and non-nitrogenous compounds (Table X I I ) . T h e coupled decomposition of amino acids is commonly referred t o as t h e Stickland reaction. T h e characteristic feature of t h e Stickland reaction is t h a t single amino acids are not decomposed appreciably, b u t appropriate pairs of amino acids are decomposed rapidly.

One member of t h e pair is oxidized while t h e other is reduced.

Evidence for this formulation was obtained initially b y studying t h e interaction between amino acids and suitable redox dyes in t h e presence of cell suspensions of C. sporogenes* Alanine and some other amino acids were found t o serve as substrates for t h e reduction of methylene blue. This indicated t h a t these amino acids were oxidized. Glycine, proline, a n d h y ­ droxyproline were ineffective for reducing methylene blue, b u t rapidly oxi­

dized t h e reduced form of t h e low potential dye, benzyl viologen. This indi­

cated t h a t these amino acids were reduced. When one mole of t h e oxidizable amino acid alanine was combined with two moles of t h e reducible amino acid glycine, three moles of ammonia were formed rapidly in t h e presence of a cell suspension. This showed t h a t both amino acids were deaminated.

However, when one mole of alanine was incubated with an excess of proline only one mole of ammonia was formed. This suggested t h a t proline was reduced b y ring cleavage according to reaction (47) without formation of ammonia.

T h e correctness of this suggestion was established b y identifying

δ-amino-valerate as a major product of t h e reaction.^{1 5 2} Once t h e product of proline reduction was identified, t h e other compounds formed in the coupled decom­

position of alanine and proline could be recognized as products of alanine oxidation.⁴ Quantitative analysis showed t h a t one mole each of ammonia, carbon dioxide, and acetate were formed per mole of alanine added, accord­

ing to the equation:

C H⁸C H N H²C O O H + 2 H²0 N H³ + C H³C O O H + C 0² (48) When one mole of alanine was incubated with an excess of glycine, three moles of ammonia, three moles of acetate, and one mole of carbon dioxide were formed. On t h e assumption t h a t t h e same products were formed from alanine in the presence of glycine or proline, these d a t a lead to t h e conclu­

sion t h a t glycine is reduced to acetate and ammonia [equation (49)].

CH²NH²COOH + 2H -» N H , + CH³COOH (49) T h e reduction of glycine to acetate and ammonia was subsequently con­

firmed by experiments which were based upon t h e ability of some strains of C. sporogenes to use gaseous hydrogen as a reducing agent.^{1 2} When a cell suspension was incubated with a known amount of glycine in a hydrogen atmosphere, the quantity of hydrogen consumed and products formed agreed well with equation (49).

Tracer experiments^{1 5 3} have shown t h a t the carboxyl and methyl carbon atoms of acetate are derived from the carboxyl and methylene carbon atoms of glycine, respectively. Two moles of glycine are necessary to oxidize one mole of alanine [equation (50)].

alanine + 2 glycine + 2 H²0 -> 3 N H³ + 3CH³COOH + C 0² (50) A. OXIDATIONS

A number of other amino acids can serve as reductants for Clostridia catalyzing the Stickland reaction (see Table X I I I ) . These amino acids m a y be divided into three groups⁵: (1) Aliphatic amino acids t h a t are more reduced t h a n α-keto acids, namely, alanine, leucine, isoleucine, norleucine, and valine. (2) Aliphatic amino acids t h a t are in the same oxidation state as the keto acids, namely, serine, threonine, cysteine, methionine, arginine, citrulline, and ornithine. (3) Other amino acids, including histidine, phenyl­

alanine, tryptophan, tyrosine, aspartate, and possibly glutamate. N o t all of these amino acids are used by any one species and the rates of oxidation of the different amino acids by one organism m a y differ widely.

Clostridium sporogenes oxidizes amino acids of group 1 rapidly and with about equal facility, as judged by the rate of reduction of brilliant cresyl blue. Amino acids of the other groups are used at lower rates or not at all

T A B L E X I I I

AMINO ACIDS USED AS REDUCTANTS IN THE STICKLAND REACTION

In document Fermentations of Nitrogenous Organic Compounds (Pldal 42-47)