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Evolution of the bacterial phosphotransferase
system: from carriers and enzymes to
group translocators
M.H. Saier, Jr1 , R.N. Hvorup and R.D. Barabote
Division of Biological Sciences, University of California at San Diego, La Jolla, CA 92093-0116, U.S.A.
Abstract
The bacterial phosphotransferase system (PTS) is a structurally and functionally complex system with a surprising evolutionary history. The substrate-recognizing protein constituents of the PTS (Enzymes II) derive
from at least four independent sources. Some of the non-PTS precursor constituents have been identified,
and evolutionary pathways taken have been proposed. Our analyses suggest that two of these independently
evolving systems are still in transition, not yet having acquired the full-fledged characteristics of PTS
Enzyme II complexes. The work described provides detailed insight into the process of catalytic protein
evolution.
Introduction
For the past 30 years, our laboratory has been interested in
various aspects of transmembrane molecular transport [1,2].
For the purpose of understanding these processes, we have
applied both bioinformatic and molecular biological approaches [3,4]. As part of our research efforts, we have classified transport proteins into a unified transporter classification
(TC) system [5,6] (see the TCDB database at http://saier144-164.ucsd.edu/tcdb/), and have identified homologues of
these proteins in fully sequenced genomes of both procaryotes and eukaryotes [7,8]. We have used bioinformatic
approaches to answer fundamental questions about transport
[9] and to trace pathways of transport protein evolution [10].
In this brief article, we will focus on the fascinating story
underlying the evolution of the bacterial phosphotransferase
system (PTS). This system is incredibly complex, both from
functional (Table 1) and structural (Figure 1) standpoints [11–
16]. In a single bacterium, up to 3.2% of all the genes within
the organism’s genome may encode the proteins of this system
(R.D. Barabote and M.H. Saier, unpublished work).
Most of the substrate-specific PTS enzymes to be discussed
in this article are derived from Escherichia coli [17]. However,
it should be kept in mind that the diversity of the PTS is far
greater than can be found in any one organism. Much of this
diversity, recognized on the basis of whole bacterial genome
analyses, is yet to be discovered.
Families of PTS permeases
Group translocators of the PTS transport and phosphorylate
their sugar substrates in a single concerted process. The phosKey words: energy coupling, evolution, phosphotransferase system, secondary carriers,
transport.
Abbreviations used: Asc, ascorbate; Chb, diacetylchitobiose; Dha, dihydroxyacetone; Fru,
Fructose; Gat, Galactitol; Glc, glucose; Glc’d, glucoside; Lac, lactose; MFS, major facilitator
superfamily; Man, Mannose; Mtl, mannitol; PEP, phosphoenolpyruvate; PTS, phosphotransferase
system; TC, transporter classification; TMS, transmembrane spanner.
1
To whom correspondence should be addressed (email [email protected]).
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phoryl group that allows energy coupling to transport is
derived from the end product of glycolysis, phosphoenolpyruvate (PEP). The protein constituents of the system are
depicted in Figure 1. Enzyme I and HPr are the pathwayspecific energy-coupling proteins of the PTS. These proteins
serve to phosphorylate the family-specific PTS energycoupling proteins, the Enzymes IIA, which in turn transfer
their phosphoryl groups to the Enzymes IIB. The latter enzymes are the permease-specific PTS phosphoryl donors for
sugar uptake and phosphorylation. Of all the PTS constituents, only the integral membrane Enzyme IIC permeases are
not phosphorylated (Figure 1). The IIC proteins provide the
basis for classification of PTS permeases in the category 4.A
of the TC system [5,6].
At least four evolutionarily distinct (super)families of
PTS Enzyme II complexes are currently recognized. These
families are (1) the Glucose (Glc)-Fructose (Fru)-Lactose
(Lac) superfamily, (2) the Ascorbate (Asc)-Galactitol (Gat)
superfamily, (3) the Mannose (Man) family and (4) the Dihydroxyacetone (Dha) family. The origin of the IIC constituents of the Glc-Fru-Lac superfamily with 6–8 α-helical
transmembrane spanners (TMSs) is not known, but the IIC
permeases of the Asc-Gat superfamily have 12 putative TMSs
and are believed to have arisen from 12 TMS permeases of the
major facilitator superfamily (MFS; TC #2.A.1). Moreover,
the IIC constituents of the Man family may have arisen from
primordial 6 TMS permeases similar to sugar-transporting
ABC systems (TC #3.A.1.2), and the water soluble, nontransporting Dha family PTS Enzyme II complexes with
0 TMSs arose from cytoplasmic ATP-dependent Dha kinases.
The Glc-Fru-Lac superfamily
Table 2, part A summarizes some of our thoughts about the
origins of the PTS permeases of the Glc-Fru-Lac superfamily.
We have proposed that the primordial PTS was specific for
D-fructose [18]. This is the only sugar that feeds directly
into glycolysis without rearrangement of its carbon backbone
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Table 1 The PTS: functional complexity
Property no.
Function
1
Chemoreception
2
3
Transport
Sugar phosphorylation
4
5
6
Protein phosphorylation
Regulation of non-PTS transport
Regulation of carbon metabolism
7
8
9
Coordination of nitrogen and carbon metabolism
Regulation of gene expression
Regulation of pathogenesis
10
Regulation of cell physiology
Figure 1 Schematic depiction of the protein constituents of a
typical PTS permease
A PTS permease is a sugar transporting Enzyme II complex of the bacterial
PEP-dependent phosphotransferase system. The sugar substrate (S) is
transported from the extracellular medium through the membrane in a
pathway determined by the integral membrane permease-like Enzyme
IIC (C) constituent, often a homodimer in the membrane as shown.
The sequentially acting energy-coupling proteins transfer a phosphoryl
group from the initial phosphoryl donor, phosphoenolpyruvate (PEP),
to the ultimate phosphoryl acceptor, sugar, yielding a sugar-phosphate
(S-P). These enzymes are: Enzyme I (I), HPr (H), Enzyme IIA (A) and
Enzyme IIB (B). I is the first general energy-coupling protein; H is the
second general energy-coupling protein; A is the indirect family-specific
phosphoryl donor; B is the direct permease-specific phosphoryl donor;
and C is the permease/receptor that energizes transport of the sugar
substrate. A given bacterial cell may possess multiple PTS Enzyme II
complexes, each specific for a different set of sugars. Some bacteria
also possess multiple sets of PTS energy-coupling proteins (Enzymes I,
HPr, IIA and IIB) that may play regulatory roles independently of sugar
transport.
skeleton. Moreover, many bacteria possess fructose-specific
PTS permeases but lack all others. If glycolysis was, in fact,
the original sugar metabolic pathway, it follows that fructose
may have been the first sugar to provide energy via glycolysis
to primordial cells.
Most closely related to the fructose-specific PTS permeases
are the mannitol (Mtl)-specific systems. However, more
distantly related are many that transport glucose (Glc) and its
derivatives such as N-acetyl glucosamine as well as a variety
of glucosides (Glc’d) including maltose, trehalose and various β-glucosides (Figure 2). The Glc and Glc’d types are
more closely related to each other than they are to the Fru/Mtl
permeases. Finally, in an even more divergent subfamily
of the Glc-Fru-Lac superfamily are PTS permeases specific
for lactose (Lac) and diacetylchitobiose (Chb) (Figure 2).
Interestingly, although all of these IIC proteins/domains are
demonstrably homologous [19], this is clearly not the case
for the IIA and IIB proteins that provide energy coupling for
transport and allow substrate sugar phosphorylation.
For example, high-resolution three-dimensional X-ray structures are available for several of these proteins, and IIAGlc
has an entirely different fold than is observed for the E. coli
IIAMtl or the lactose-specific IIA protein, IIALac . Moreover, IIBGlc has a very different 3D structure from IIBChb
(Table 2, part A). We therefore conclude that the coupling
of phosphoryl transfer to transport as catalysed by IIC
permeases of the Glc-Fru-Lac superfamily resulted from the
superimposition of structurally distinct sets of functionally
equivalent phosphoryl transfer proteins onto the transporters.
The Asc-Gat superfamily
Properties of L-ascorbate (Asc)-D-galactitol (Gat) superfamily permeases are summarized in Table 2, part B. These
systems have only recently been characterized from genetic,
biochemical and phylogenetic standpoints [4,20–22]. We
believe that these PTS permeases arose from secondary
carriers of the major facilitator superfamily (MFS). Hvorup
et al. [20] have presented the evidence for this claim.
The Asc-Gat superfamily consists of two subfamilies
that exhibit distinctive properties. Although all of the IIC
constituents of this superfamily exhibit 12 putative TMSs and
presumably arose from MFS carriers, our analyses revealed
that IICAsc homologues are frequently fused to their IIA and
IIB energy-coupling proteins although surprisingly, this is
never true of IICGat homologues (Table 2, part B). Moreover,
homologues of IICAsc are always encoded in operons with
genes encoding IIA and IIB proteins, suggesting a tight
functional association, but this is not true of IICGat -encoding
genes that are often present in operons that do not encode
either IIA or IIB or both. Finally, some IICGat homologues
are encoded in the genomes of organisms that lack genes for
all other PTS proteins including Enzyme I and HPr as well
as Enzymes IIA and IIB. This last observation means that
these IICGat homologues cannot function in these bacteria
by a PTS-type mechanism. Although none of these putative
transporters is yet functionally characterized, we believe
that they must function as secondary carriers. Because Asc
and Gat IIA and IIB proteins are distantly related to the
corresponding proteins of the Glc-Fru-Lac superfamily, we
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Biochemical Society Transactions (2005) Volume 33, part 1
Table 2 Evolutionarily relevant characteristics of four families of PTS enzyme II complexes
Enzyme family/property no.
Characteristic
A) The Glc-Fru-Lac superfamily
1
2
Fru: The original PTS (proposed)
Proposed evolutionary pathway as depicted in Figure 2.
3
Mosaic origins of IIAs and IIBs:
IIAGlc is not homologous to IIAMtl , IIANtr or IIALac
IIBGlc is not homologous to IIBChb
B) The Asc-Gat superfamily
1
2
IICAsc homologues are often fused to IIA and IIB homologues, but IICGat homologues never are
IICAsc homologues are always encoded by genes in operons with IIA and IIB genes, but IICGat
homologues can be encoded in operons lacking IIA and IIB genes
Some IICGat homologues are found in organisms that lack all other PTS proteins
Asc and Gat IIA and IIB constituents are distantly related to IIA and IIB constituents of the
3
4
Glc-Fru-Lac superfamily
C) The Man family
1
2
3
D) The Dha family
All constituents (IIA, IIB, IIC and IID) differ structurally from all other PTS permease proteins
All members, but only members of this family, have IID constituents
The IIB constituents are phosphorylated on His rather than Cys
1
2
DhaK and DhaL correspond to the N- and C-termini of ATP-dependent DHA kinases
DhaM consists of three domains: IIAMan -HPr-I
3
The three domains of DhaM are phosphorylated by PEP, EI and HPr, but DhaK and L are not
phosphorylated
DhaK binds DHA covalently to a His residue and transfers the phosphoryl group from IIA of DhaM
4
via DhaL-ADP to DHA
Figure 2 Proposed pathway for the evolution of currently
recognized subfamilies of the Glc-Fru-Lac superfamily from a
primordial Fructose (Fru) Enzyme II permease complex
PTS phosphoryl transfer energy coupling may not have yet
evolved so as to be obligatory. Gat family permeases may still
be in transition, not yet having acquired all of the functional
characteristics of full-fledged PTS permeases.
The Man family
can presume that inclusion of these proteins in the PTS
Enzyme II complexes of the Asc-Gat superfamily resulted
from the superimposition of the pre-existing IIA and IIB
phosphoryl transfer proteins onto MFS-like carriers. However, in contrast to Asc family members, Gat family members may be promiscuous, being capable of functioning
both as secondary carriers and as PTS permeases. Thus,
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We previously called the Man family of PTS permeases the
‘splinter group’ family because of their distinctive properties
(Table 2, part C). These systems catalyse the transport and
coupled phosphorylation of a variety of hexoses including
mannose, glucose, glucosamine, fructose, galactosamine and
N-acetylgalactosamine. All four constituents of these systems
(IIA, IIB, IIC and IID) differ structurally from the constituents of the PTS permeases of all other families. Moreover,
only this family has a IID constituent, and the IIB enzymes
accept the phosphoryl group from the IIA constituent on a
histidyl residue rather than a cysteyl residue as is true of all
other IIB constituents of PTS permease complexes. It seems
clear that not only the IIC constituents, but also all four
protein domains of the Man family permeases (IIA–IID)
arose independently of all other PTS Enzyme II complex
constituents.
The Dha family
Early evidence suggested that in E. coli, dihydroxyacetone
(DHA) might be phosphorylated in a PTS-dependent
Transporters 2004
mechanism [23]. When the E. coli genome sequence became
available for analysis, a tricistronic dha operon was identified,
including one gene encoding a multidomain PTS protein with
a structure different from any reported previously, as well as
two ‘split’ genes that showed homology throughout their
combined lengths with ATP-dependent DHA kinases from
other bacteria [7,24]. The properties of these three proteins,
which together comprise an unusual, cytoplasmic Enzyme II
complex, are summarized in Table 2, part D. DhaK and
DhaL, encoded by the latter two genes, correspond to the Nand C-terminal domains of the homologous ATP-dependent
DHA kinases. DhaK binds DHA covalently to a histidyl
residue in the protein (providing strict specificity) whereas,
DhaL, which includes the nucleotide-binding region of DHA
kinases, contains tightly bound ADP [25]. The three-domain
DhaM protein consists of an N-terminal IIAMan -like domain, a central HPr-like domain and a truncated Enzyme I
domain [26]. All three domains can be phosphorylated
directly using PEP as the phosphoryl donor in the presence
of authentic Enzyme I and HPr. This phosphorylated DhaM
protein can then transfer its phosphoryl group to the tightly
bound ADP in DhaL which can then phosphorylate DHA in
the presence of DhaK [27]. Thus, DhaK is the functional
equivalent of a typical PTS Enzyme IIC, DhaL is the
functional equivalent of a typical PTS Enzyme IIB and DhaM
is the functional equivalent of a typical PTS Enzyme IIA. The
system retains some of the features of the ATP-dependent
kinase of origin, but it has acquired some of the unique
properties of a PTS Enzyme II complex [26]. Like the Gat
family discussed in the previous section, the DHA family of
Enzyme II complexes seems to be in transition. However,
in contrast to Gat family permeases, and unlike all IIB
constituents of PTS permeases, DhaL is not phosphorylated
on an amino acyl residue and instead retains the essential
cofactor, ADP, that served as the phosphoryl doning substrate
in the precursor ATP-dependent DHA kinase. Tightly bound
ADP thus provides the function of the active site His or Cys
residue in other IIB enzymes.
Concluding remarks
Examination of the PTS reveals the results of complex evolutionary processes at various apparent stages of completion.
Well-characterized PTS permeases of the Glc-Fru-Lac and
Man families appear to be well integrated into the PTS
machinery; they cannot transport sugars by any mechanism
other than the PTS-dependent group translocation mechanism. They only transport and phosphorylate their substrates
via the typical, PTS-type phosphoryl transfer chain, exclusively using protein residue phosphorylation without
the need for organic cofactors.
The Asc-Gat superfamily seems to have one well-integrated family of PTS permeases (Asc) that can only function
by a PTS-dependent mechanism, but the IIC constituents of
the other family (Gat) seem to be capable of a more promiscuous existence. They may be able to function either as
secondary carriers or as PTS/phosphoryl transfer-dependent
group translocators, depending on the specific system. It
is also possible that some of these IIC constituents of the
Gat family can function by both mechanisms, depending on
the availability of the energy-coupling phosphoryl transfer
proteins.
PTS Enzyme II complexes of the Dha family show
close sequence similarity with their ATP-dependent DHA
kinase homologues, and the recent elegant biochemical and
structural analyses conducted in the Erni laboratory [24–26]
have revealed mechanistic similarities as well. Most strikingly,
DhaL retains an organic ‘cofactor’, tightly bound ADP, and
this cofactor must be phosphorylated at the expense of PEP
in order to complete phosphoryl transfer to DHA. Thus,
the DHA Enzyme II complex exhibits mechanistic features
common to both its ATP-dependent evolutionary precursor
enzyme and the PEP-dependent enzyme complex into which
it is in the process of evolving. Seldom do we get such a vivid
snapshot of evolution in transition.
The observations reported here confirm the postulate that
the PTS is a recently evolving system, a postulate that was originally based on its exclusive presence in bacteria. No recognized homologues of PTS proteins in eukaryotes or archaea
have been found (R. Barabote and M.H. Saier, Jr, unpublished
work).
We want to acknowledge the important contributions of several
investigators of the PTS, particularly Bernhard Erni and Joseph
Lengeler who respectively have conducted detailed analyses of the
Dha and Gat PTS Enzyme II complexes of E. coli. We also acknowledge the important contributions of many students, postdoctoral
fellows and visiting scholars in the Saier laboratory. We thank Mary
Beth Hiller for assistance in the preparation of this manuscript. This
work was supported by NIH grant GM64368 from the National
Institute of General Medical Sciences.
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