The mechanism of utilization of polyphosphate by polyphosphate glucokinase from Propionibacterium shermanii

The phosphorylation of glucose by polyphosphate glucokinase with both long- and short-chain polyphosphates has been shown to occur by either a nonprocessive mechanism, i.e. with repeated association and dissociation of the polyphosphate from the enzyme after each phosphorylation or by a quasiprocessive mechanism in which several phosphorylations occur prior to the release of polyphosphate and the reassociation with the enzyme. In contrast, the phosphorylation of ADP to ATP by polyphosphate kinase is by a strictly processive mechanism; the phosphorylation occurs without release of the polymer from the enzyme prior to termination of the reaction (Robinson, N. A., Clark, J. E., and Wood, H. G. (1987) J. Biol. Chem. 262, 5216-5222). The demonstration that the mechanism is quasi-or nonprocessive was accomplished by electrophoresis using a variety of concentrations of polyacrylamide gels which made it possible to detect the intermediate sizes formed during the reactions. It also has been shown that all chains longer than about 100 are used simultaneously, but with chains of less than 100 residues, there is preferential utilization of the longest chains. Thus a narrow range of sizes is formed from a heterogeneous mixture of long chains. It is this formation of the narrow range of sizes that makes it possible to use polyphosphate glucokinase for the determination of the average size of long chains (Pepin, C. A., Wood, H. G., and Robinson, N. A. (1986) Biochem. Int. 12, 111-123).     

Isolation and characterization of a pyrophosphate-dependent phosphofructokinase from Propionibacterium shermanii.

A pyrophosphate-dependent phosphofructokinase (pyrophosphate; D-fructose-6-phosphate-1-phosphotransferase) has been purified and characterized from extracts of Propionibacterium shermanii. The enzyme catalyzes the transfer of phosphate from pyrophosphate to fructose 6-phosphate to yield fructose-1,6-P2 and phosphate. This unique enzymatic activity was observed initially in Entamoeba histolytica (Reeves, R.E., South, D.J., Blytt, H.G., and Warren, L. G. (1974) J. Biol. Chem. 249, 7734-7741). This is the third pyrophosphate-utilizing enzyme that these two diverse organisms have in common. The others are phosphoenolpyruvate carboxytransphosphorylase and pyruvate phosphate dikinase. The PPi-phosphofructokinase from P. shermanii is specific for fructose-6-P and fructose-1,6-P2, no other phosphorylated sugars were utilized. Phosphate could be replaced by arsenate. The Km values are: phosphate, 6.0 X 10(-4) M; fructose-1, 6-P2, 5.1 X 10(-5) M; pyrophosphate, 6.9 X 10(-5) M; and fructose-6-P, 1.0 X 10(-4) M. The S20w is 5.1 S. The molecular weight of the native enzyme is 95,000. Sodium dodecyl sulfate electrophoresis of the enzyme showed a single band migrating with an Rf corresponding to a molecular weight of 48,000. Extracts of P. shermanii have PPi-phosphofructokinase activity approximately 6 times greater than ATP-phosphofructokinase and 15 to 20 times greater than fructose diphosphatase activities. It is proposed that (a) PPi may replace ATP in the formation of fructose-1-6-P2 when the organism is grown on glucose and (b) when the organism is grown on lactate or glycerol the conversion of fructose-1,6-P2 to fructose-6-P during gluconeogenesis may occur by phosphorolysis rather than hydrolysis.     

13C-NMR study of lactate metabolism in Propionibacterium freudenreichii subsp. shermanii

The adaptation to osmotic stress in Propionibacterium freudenreichii subsp. shermanii was investigated by using natural-abundance ¹³C nuclear magnetic resonance spectroscopy. Cells incubated either in a standard laboratory medium or in a medium designed to simulate the physicochemical conditions of Swiss-type cheese were found to accumulate different levels of osmotic-stress-protectant molecules. Proline, betaine, trehalose and glutamate were found simultaneously. Moreover, two types of polysaccharides were in evidence in this strain. Lactate catabolism was not mainly directed towards cell growth requirements and organic acid production but also towards biosynthesis of osmolytes requested for adaptation in a cheese environment. The possible involvement of such type of metabolite accumulation in the main cheese-ripening bacteria in Swiss-type cheeses is discussed.     

Regulation of pyruvate kinase from Propionibacterium shermanii

Pyruvate kinase from Propionibacterium shermanii was shown to be activated by glucose-6-phosphate (G-6-P) at non-saturating phosphoenol pyruvate (PEP) concentrations but other glycolytic and hexose monophosphate pathway intermediates and AMP were without effect. Half-maximal activation was obtained at 1 mM G-6-P. The presence of G-6-P decreased both the PEP_0.5V and ADP_0.5V values and the slope of the Hill plots for both substrates. The enzyme was strongly inhibited by ATP and inorganic phosphate (P_i) at all PEP concentrations. At non-saturating (0.5 mM) PEP, half-maximal inhibition was obtained at 1.8 mM ATP or 1.4 mM P_i. The inhibition by both P_i and ATP was largely overcome by 4 mM G-6-P. The specific activity of pyruvate kinase was considerably higher in lactate-, glucose- and glycerol-grown cultures than that of the enzyme catalysing the reverse reaction, pyruvate, phosphate dikinase. It is suggested that the activity of pyruvate kinase in vivo is determined by the balance between activators and inhibitors such that it is inhibited during gluconeogenesis while, during glycolysis, the inhibition is relieved by G-6-P.Abbreviations PEP phosphoenolpyruvate - G-6-P glucose-6-phosphate - P_i inorganic phosphate     

Presence of lactate dehydrogenase and lactate racemase in Megasphaera elsdenii grown on glucose or lactate.

Activity of D-lactate dehydrogenase (D-LDH) was shown not only in cell extracts from Megasphaera elsdenii grown on DL-lactate, but also in cell extracts from glucose-grown cells, although glucose-grown cells contained approximately half as much D-LDH as DL-lactate-grown cells. This indicates that the D-LDH of M. elsdenii is a constitutive enzyme. However, lactate racemase (LR) activity was present in DL-lactate-grown cells, but was not detected in glucose-grown cells, suggesting that LR is induced by lactate. Acetate, propionate, and butyrate were produced similarly from both D- and L-lactate, indicating that LR can be induced by both D- and L-lactate. These results suggest that the primary reason for the inability of M. elsdenii to produce propionate from glucose is that cells fermenting glucose do not synthesize LR, which is induced by lactate.     

Propionic acid fermentation of glycerol and glucose by Propionibacterium acidipropionici and Propionibacterium freudenreichii ssp.shermanii

A comparative study was carried out in anaerobic batch cultures on 20 g/l of either glycerol or glucose using two propionibacteria strains, Propionibacterium acidipropionici and Propionibacterium freudenreichii ssp. shermanii. In all cases, fermentation end-products were the same and consisted of propionic acid as the major product, acetic acid as the main by-product and two minor metabolites, n-propanol and succinic acid. Evidence was provided that greater production of propionic acid by propionibacteria was obtained with glycerol as carbon and energy sources. P. acidipropionici showed higher efficiency in glycerol conversion to propionic acid with a faster substrate consumption (0.64 g l⁻¹ h⁻¹) and a higher propionic acid production (0.42 g l⁻¹ h⁻¹ and 0.79 mol/mol). The almost exclusive production of propionic acid from glycerol by this bacterium suggested an homopropionic tendency of this fermentation. Acetic acid final concentration was two times lower on glycerol (2 g/l) than on glucose (4 g/l) for both micro-organisms. P. freudenreichii ssp. shermanii exhibited a glycerol fermentation pattern typical of non-associated glycerol-consumption-product formation. This could indicate a particular metabolism for P. freudenreichii ssp. shermanii oriented towards the production of other specific components. These results tend to show that glycerol could be an excellent alternative to conventional carbon sources such as carbohydrates for propionic acid production. Received: 21 May 1999 / Accepted: 1 November 1999     

Polyphosphate kinase from Propionibacterium shermanii. Demonstration that the synthesis and utilization of polyphosphate is by a processive mechanism

The mechanism of synthesis of inorganic polyphosphate by polyphosphate kinase (EC 2.7.4.1) from Propionibacterium shermanii is shown to be processive. Analysis of the synthesized polyphosphate on polyacrylamide gels, which resolve on the basis of molecular weight, proves that the elongation reaction occurs without dissociation of intermediate sizes of the polymer from the enzyme. As a consequence, only high molecular weight polyphosphates are synthesized. The mechanism is processive both in the presence and absence of basic protein. It has been shown previously that basic proteins stimulate the synthesis of polyphosphate (Robinson, N.A., Goss, N.H., and Wood, H.G. (1984) Biochem. Int. 8, 757-769). In addition, using a similar method, it is shown that the reverse reaction, the utilization of polyphosphate to phosphorylate ADP, occurs by a processive mechanism. Accordingly, polyphosphates formed by polyphosphate kinase in the cell would be entirely high molecular weight.     

Polyphosphate kinase from Propionibacterium shermanii. Demonstration that polyphosphates are primers and determination of the size of the synthesized polyphosphate

Polyphosphate kinase from Propionibacterium shermanii was purified to 70% homogeneity and shown to be a monomeric enzyme of molecular weight 83,000 +/- 3,000. It was demonstrated that short chains of polyphosphate serve as primers by using [32P]polyphosphate, 6-80 residues in length for synthesis of long-chain polyphosphate glucokinase, the radiolabel was found to be at the end of the polymer, proving that the mechanism of elongation of polyphosphate by polyphosphate kinase is strictly processive. Only 1 out of 3-8 of the polyphosphate chains contained the primer, indicating that there is a second unknown pathway of initiation which does not involve the polyphosphate primer. The termination of polyphosphate synthesis was investigated. With polyphosphate as a primer, the majority of the synthesized polyphosphate was 750 residues in length. With phosphate, in place of the polyphosphate primer, the major portion was about 2,000 residues in length but there was a large span of chain lengths down to 300. Termination is influenced by pH, temperature, and the concentration of the polyphosphate primer, with the chain length decreasing as either the temperature or the concentration of primer is increased.     

X-ray structure of the cambialistic superoxide dismutase from Propionibacterium shermanii active with Fe or Mn

The first structure of a cambialistic superoxide dismutase (SOD) from Propionibacterium shermanii exhibiting similar activity with iron and with manganese was solved at a resolution of 1.6?Å and 1.9?Å respectively. Surprisingly, no obvious differences between the two SODs were observable. The protein crystallises as a homo dimer in the asymmetric unit. Because of the crystallographic symmetry, it forms a tetramer. Structures of both the manganese and the ferric form were solved using molecular replacement techniques and multiple isomorphous replacement. The tertiary structure is similar to that of the other superoxide dismutases, the metal being fivefold coordinated by three histidines, one aspartate and one water molecule. The second shell of residues consists of hydrophobic amino acids, histidines and two water molecules, which are assumed to be involved in both the catalytic activity and structural stability of this superoxide dismutase. This shell may also be responsible for the cambialistic behaviour. This work shows that the reason for the metal specificity is not trivial, although minor alterations in the metal environment might be responsible for this behaviour.     

Polyphosphate kinase from Propionibacterium shermanii: formation of an enzymatically active insoluble complex with basic proteins and characterization of synthesized polyphosphate

Polyphosphate kinase, which catalyzes the synthesis of polyphosphate from ATP, has been partially purified from Propionibacterium shermanii. The reaction is unusual in that addition of basic protein causes the enzyme to precipitate and the insoluble form has optimal activity. The synthesized [32P]polyphosphate is non-covalently bound to the precipitated material and was isolated from the complex by proteolysis. The gel electrophoresis procedure of Maxam and Gilbert was adapted to sizing polyphosphates. When polyphosphate was treated with alkali, polyphosphates ranging from 1-100 phosphate residues were obtained as individual bands. The untreated enzymatically synthesized polyphosphate migrated as a species in excess of 200 phosphate moieties.     

Purification and characterization of methylmalonyl-CoA epimerase from Propionibacterium shermanii.

Methylmalonyl-CoA epimerase, which specifically interconverts the (2R)- and (2S)- epimers of methylmalonyl-CoA, was purified 95-fold from Propionibacterium shermanii by a new method that affords apparently homogeneous enzyme, in 80-100mg quantities, in yields representing about 40% of the activity in cell-free extracts. The specific activity of the purified enzyme, 10.1 mukat/mg, is much greater than previously reported. Native methylmalonyl-CoA epimerase has Mr about 33000, and apparently consists of two identical subunits. The purified enzyme is stable indefinitely when stored at -20 degrees C and pH 8.5, but contrary to previous reports it is not unusually acid-stable. The activity of methylmalonyl-CoA epimerase is increased by Co2+, and to a smaller extent by Ni2+, Mn2+ and Zn2+.     

Purification of methylmalonyl-CoA mutase from Propionibacterium shermanii using affinity chromatography.

A novel procedure for the purification of methylmalonyl-CA mutase from Propionibacterium shermanii has been described which employs affinity chromatography on a column of immobilized vitamin B-12 linked covalently to Sepharose. The method has the advantage of being simple and rapid, thus enabling the purification of the enzyme to near homogeneity with good yields.