Page 93 - The Biochemistry of Inorganic Polyphosphates

March 9, 2004
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Enzymes of polyphosphate degradation 77
The purified exopolyphosphatases from E. coli and A. johnsonii have low specific activi-
ties (1 µmol P i per min per mg protein for the enzyme from A. johnsonii and 22 µmol P i per
min per mg of protein for the enzyme from E. coli) in comparison with the yeast enzymes
(200–400 µmol P i per min per mg of protein and more). They are low-active with PolyP 3
and short-chain PolyPs and require K for the maximal activity. These properties represent
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the most appreciable difference between the majority of yeast and bacterial exopolyphos-
phatases. The low activity of bacterial exopolyphosphatases is probably explained by the
fact that polyphosphate kinase in procaryotes is able both to synthesize and to hydrolyse
PolyP (Kornberg et al., 1999). The gene ppx encoding the major E. coli exopolyphosphatase
has been cloned and sequenced (Akiyama et al., 1993). The ppx genes were cloned and se-
quenced from Pseudomonas aeruginosa (Miyake et al., 1999) andVibrio cholerae (Ogawa
et al., 2000b). It should be noted that in E. coli (Akiyama et al., 1993) and Vibrio cholerae
(Ogawa et al., 2000b) ppk1 and ppx are in one operon, which suggests a co-regulation of
their transcription activities, while in Pseudomonas aeruginosa the ppx is located in the
opposite direction from the ppk gene and they do not constitute an operon (Miyake et al.,
1999).
Another enzyme encoded by the gppA gene and possessing exopolyphosphatase activity
was purified from E. coli (Keasling et al., 1993). This enzyme is a dimer with a monomer
molecular mass of 50 kDa; K m is 0.5 nM for PolyP 500 . It has a preference for long-chain
polyPs, but one of its substrates is guanosine pentaphosphate (pppGpp), an important second
messenger in bacteria.
One cannot exclude, however, that some bacteria possess other enzymes, which can
split PolyPs. The acid phosphatase of E. coli was demonstrated to split long-chain PolyPs
with a high specific activity (190 µmol P i per min per mg of protein). The enzyme is
active without divalent cations, but has the optimal pH of 2.5 (Dassa and Boquet, 1981).
Two exopolyphosphatases have been detected in a cell-free extract of Microlunatus phos-
phovorus (Lichko et al., 2002a),a bacterium isolated from activated sludge. One of them
has a molecular mass of 93 kDa, pH optimum of 4.5, does not require K for its activ-
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ity and is stimulated by divalent cations. The other exopolyphosphatase has a molecular
mass of 55 kDa, pH optimum of 7.5, and displays its optimal activity in the presence of
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K and divalent cations. The content of the former exopolyphosphatase increased dur-
ing the growth, while that of the latter varied only slightly (Lichko et al., 2002a). Ex-
opolyphosphatase activity was found in some methanotrophs (Trotsenko and Shishkina,
1990).
There are little data on exopolyphosphatase activity in Archae. In Halobacterium sali-
narium, it was very low (Andreeva et al., 2000). In a crude extract of Sulfolobus sol-
factaricus (Cardona et al., 2002), the specific activity was 0.6 nmol P i per min per mg
of protein. This is much less than that found in bacteria. In this termophilic archae, a
functionally active gene of exopolyphosphatase was found, cloned and overexpressed.
This gene encoded a protein of 417 amino acid residues (47.9 kDa). Purified recom-
binant exopolyphosphatase degraded long-chain PolyPs (700–800 residues) and needed
Mn 2+ for its activity. The deduced amino acid sequence of S. solfactaricus ppx showed
the highest (25–45 %) similarity to the sequences of bacterial ppx and possessed all of
their conserved motifs. While in vitro the enzyme splits pppGpp, the authors believe
that Archae do not seem to possess the genes responsible for the pppGpp synthesis and
that the role of exopolyphosphatase in vivo is only PolyP hydrolysis (Cardona et al.,
2002).