Page 82 - The Biochemistry of Inorganic Polyphosphates

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Enzymes of polyphosphate biosynthesis and degradation
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can use PolyP as a donor instead of ATP, thereby converting GDP and other nucleoside
diphosphates to nucleotide triphosphates (Kuroda and Kornberg, 1997). This reaction was
observed in crude membrane fractions of E. coli and Pseudomonas aeruginosa, as well as
in purified enzyme preparations obtained from E. coli. Membrane fractions obtained from
E. coli mutants lacking the ppk1 gene have no such activity. The substrate specificity order
was ADP > GDP > UDP, CDP; the activity with ADP was twice as high as that with GDP.
It was confirmed that polyphosphate kinase efficiently catalysed UTP regeneration in the
cyclic system of N-acetyl lactosamine synthesis (Noguchi and Shiba, 1998). This activity
of pure polyphosphate kinase was used to develop a method of oligosaccharide synthesis
(Noguchi and Shiba, 1998). Although the transfer of a phosphate from PolyP to GDP by
polyphosphate kinase to produce GTP was the predominant reaction, the enzyme also trans-

ferred a pyrophosphate group to GDP to form a linear guanosine 5 -tetraphosphate (Kim
et al., 1998). The enzyme is also capable of authophosphorylation using ATP as a phosphate
donor (Tzeng and Kornberg, 2000). The diverse functions of ppk1 in E. coli are realized by
means of different sub-unit organization. Radiation target analysis revealed that the principal
functional unit of ppk1 is a dimer, but the synthesis of linear guanosine tetraphosphate and
authophosphorylation require trimeric and tetrameric states, respectively (Tzeng and Korn-
berg, 2000). The polyphosphate kinase of Vibrio cholerae is also a homotetramer (Ogawa
et al., 2000b). It resembles the ppk1 of E. coli in size and multiple activities: processive
PolyP synthesis from ATP, nucleoside diphosphate kinase activity with ADP and GDP as
acceptors and PolyP as a donor, ppGpp synthesis from GDP, and autophosphorylation. The
most notable differences are in the kinetic parameters for ATP: K m is 0.2 and 2 mM for the
ppk1 of V. cholerae and E. coli, respectively (Ogawa et al., 2000b).
Polyphosphate kinase was purified from Acinetobacter sp. (Trelstad et al., 1999). It was
a 79 kDa monomer. In contrast to the E. coli enzyme, the polyphosphate kinase purified
from this bacterium seems to work only in the forward direction, i.e. it produces but does
not degrade PolyPs.
The ppk1 genes of E. coli (Akiyama et al., 1992), Klebsiella aerogenes (Kato et al.,
1993b), Neisseria meningitidis (Tinsley and Gotschlich, 1995), Pseudomonas aeruginosa
(Ishige et al., 1998), Acinetobacter sp. (Geissdorfer et al., 1998), V. cholerae (Ogawa
et al., 2000b), Rhodocyclus tenuis (McMahon et al., 2002) and many other bacteria (see
http://www.expasy.org) have been cloned, sequenced and characterized. The deduced amino
acid sequences of these enzymes show an extensive homology in different bacterial species
(Figure6.1)(TzengandKornberg,1998).Someconservedaminoacidresiduesareimportant
for enzymatic activity. Replacement of conserved His-441 and His-460 by either glutamine
or alanine by site-specific mutagenesis rendered an enzymatically inactive protein in E. coli
(Kumble et al., 1996).
High conservatism of the ppk1 gene structure reveals polyphosphate kinase in microor-
ganisms, microbial associates and activated sludges. Fragments of putative ppk genes were
retrieved from a pure culture of Rhodocyclus tenuis and from microorganisms of activated
sludge using PCR primers (McMahon et al., 2002). Four novel ppk homologs were found
in the sludge, and two of them (types I and II) shared a high degree of amino acid similarity
with R. tenuis ppk (86 and 87 %, respectively). Dot-blot analysis of total RNA extracted
from the sludge demonstrated the presence of the Type I ppk mRNA, indicating that this
gene is expressed during the process of phosphate removal. Inverse PCR was used to obtain
a full Type I sequence from sludge DNA, and a full-length ppk was cloned, overexpressed,
and purified to near homogeneity. The purified polyphosphate kinase has a specific activity