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://saier-144-164.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 [1116]. 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of all the genes within the organism's genome may encode the proteins of this system (R.D. Barabote and M.H. Saier, unpublished work).

Table 1
The PTS: functional complexity
Property no.Function
Chemoreception 
Transport 
Sugar phosphorylation 
Protein phosphorylation 
Regulation of non-PTS transport 
Regulation of carbon metabolism 
Coordination of nitrogen and carbon metabolism 
Regulation of gene expression 
Regulation of pathogenesis 
10 Regulation of cell physiology 
Property no.Function
Chemoreception 
Transport 
Sugar phosphorylation 
Protein phosphorylation 
Regulation of non-PTS transport 
Regulation of carbon metabolism 
Coordination of nitrogen and carbon metabolism 
Regulation of gene expression 
Regulation of pathogenesis 
10 Regulation of cell physiology 

Schematic depiction of the protein constituents of a typical PTS permease

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.

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.

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 phosphoryl 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 energy-coupling 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, non-transporting 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 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.

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 Fru: The original PTS (proposed) 
 2 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 IICAsc homologues are often fused to IIA and IIB homologues, but IICGat homologues never are 
 2 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 
 3 Some IICGat homologues are found in organisms that lack all other PTS proteins 
 4 Asc and Gat IIA and IIB constituents are distantly related to IIA and IIB constituents of the Glc-Fru-Lac superfamily 
C) The Man family  
 1 All constituents (IIA, IIB, IIC and IID) differ structurally from all other PTS permease proteins 
 2 All members, but only members of this family, have IID constituents 
 3 The IIB constituents are phosphorylated on His rather than Cys 
D) The Dha family  
 1 DhaK and DhaL correspond to the N- and C-termini of ATP-dependent DHA kinases 
 2 DhaM consists of three domains: IIAMan-HPr-IΔ 
 3 The three domains of DhaM are phosphorylated by PEP, EI and HPr, but DhaK and L are not phosphorylated 
 4 DhaK binds DHA covalently to a His residue and transfers the phosphoryl group from IIA of DhaM via DhaL-ADP to DHA 
Enzyme family/property no.Characteristic
A) The Glc-Fru-Lac superfamily  
 1 Fru: The original PTS (proposed) 
 2 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 IICAsc homologues are often fused to IIA and IIB homologues, but IICGat homologues never are 
 2 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 
 3 Some IICGat homologues are found in organisms that lack all other PTS proteins 
 4 Asc and Gat IIA and IIB constituents are distantly related to IIA and IIB constituents of the Glc-Fru-Lac superfamily 
C) The Man family  
 1 All constituents (IIA, IIB, IIC and IID) differ structurally from all other PTS permease proteins 
 2 All members, but only members of this family, have IID constituents 
 3 The IIB constituents are phosphorylated on His rather than Cys 
D) The Dha family  
 1 DhaK and DhaL correspond to the N- and C-termini of ATP-dependent DHA kinases 
 2 DhaM consists of three domains: IIAMan-HPr-IΔ 
 3 The three domains of DhaM are phosphorylated by PEP, EI and HPr, but DhaK and L are not phosphorylated 
 4 DhaK binds DHA covalently to a His residue and transfers the phosphoryl group from IIA of DhaM via DhaL-ADP to DHA 

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.

Proposed pathway for the evolution of currently recognized subfamilies of the Glc-Fru-Lac superfamily from a primordial Fructose (Fru) Enzyme II permease complex

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
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

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 [4,2022]. 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 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, 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

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 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 N- and 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 [2426] 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).

Transporters 2004: International Symposium on Membrane Transport and Transporters: Focused Meeting held at Selwyn College Cambridge, 2–5 September 2004. Edited by S.A. Baldwin (Leeds, U.K.) and P.M. Taylor (Dundee, U.K.).

Abbreviations

     
  • 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

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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