Page 92 - The Biochemistry of Inorganic Polyphosphates

WU095/Kulaev
WU095-06
Enzymes of polyphosphate biosynthesis and degradation
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The first data on this enzyme were obtained by Kitasato (1928), Ingelman and Malmgren
(1947, 1948, 1949) Krishnan (1952), Malmgren (1949, 1952) and Grossman and Lang
(1962).
Much data on the properties of exopolyphosphatases were obtained from different
sources, and have been reviewed (Kulaev, 1979; Kulaev and Vagabov 1983). The greatest
difficulty in the investigation of exopolyphosphatases was their low stabilities at purifica-
tion. The first purified preparation of an exopolyphosphatase with high activity and stability
was obtained from the S. cerevisiae cell envelope (Andreeva et al., 1990).
In recent years, the overview of the exopolyphosphatases classification and function has
been renewed a great deal. A significant diversity of exopolyphosphatases in microorgan-
isms and sufficient differences in their structure and properties in procaryotes and eucaryotes
have come to light.
The most important enzymes exhibiting exopolyphosphatase activity in bacteria are the
exopolyphosphatase encoded by the ppx gene (Akiyama et al., 1993) and the guanosine
pentaphosphate phosphohydrolase encoded by the gppA gene (Keasling et al., 1993).The
enzymes encoded by the ppx and gppA genes demonstrate a great sequence similarity,
i.e. 39 % identity over an overlapping region of 492 residues (Reizer et al., 1993). These
proteins are of about the same length (513 amino acid residues for ppx and 494 for pppGpp
phosphohydrolase). Both enzymes possess one hydrophobic region. The ppx and gppA
possess five conserved boxes, which suggest that the two phosphatases belong to the sugar
kinase/actin/heat-shock protein hsp70 superfamily (Reizer et al., 1993).
The exopolyphosphatase encoded by the ppx of E. coli is a dimer with a sub-unit molecu-
lar mass of about 58 kDa (Akiyama et al., 1993). Its affinity to high-molecular-weight PolyP
was nearly 100-fold higher than that of yeast polyphosphatases (K m = 9 nM PolyP 500 as a
polymer). This enzyme exhibits a high requirement for K (21-fold stimulation by 175 mM
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of K ) (Akiyama et al., 1993). It is ‘low-active’ with short-chain PolyPs.
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The exopolyphosphatase ppx of E. coli is a highly processive enzyme demonstrating
the ability to recognize PolyPs of long chain lengths. Multiple PolyP binding sites were
identified in distant portions of the enzyme and shown to be responsible for the enzyme
polymer length recognition (Bolesch and Keasling, 2000a). In addition, two independently
foldeddomainswereidentified.ThegenesfortheN-andC-terminaldomainsweregenerated
by using PCR and overexpressed in E. coli. The purified domain proteins were immobilized
and used for the study of PolyP binding constants. The purified N- and C-terminal domains
lacked exopolyphosphatase activity. However, the activity could be recovered in cases
where the polypeptides were combined (Bolesch and Keasling, 2000a). The N-terminal
domain contained a quasi-processive polyphosphatase active site belonging to the sugar
kinase/actin/heat-shock protein hsp70 superfamily. The C-terminal domain contained a
single polyphosphate-binding site and was responsible for nearly all affinity for PolyP. This
domain was also found to confer a highly processive mode of action (Bolesch and Keasling,
2000a).
The exopolyphosphatase encoded by ppx from A. johnsonii is a monomeric protein of
55 kDa (Bonting et al., 1993b). The K m value for a polyphosphate with an average chain
length of 64 phosphate residues is 5.9 µM. The activity is maximal in the presence of 2.5
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mM Mg 2+ and 0.1 mM K . No activity is observed in the absence of cations or in the
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presence of Mg 2+ or K alone. The enzyme of A. johnsonii was active with PolyP 3 and
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PolyP 4 in the presence of 300 mM NH 4 and 10 mM Mg , while no activity with PolyP 3
was observed in the presence of 0.1 mM K and2mMMg .
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