Fructose-bisphosphate aldolases: an evolutionary history

TIBS 1 7 - MARCH 1992 impossible at this point to assess their relationship to the better-studied systems discussed above.

Acknowledgements We thank W. W. Cieland and R. L. Van Etten for helpful comments on the manuscript, and E. E. Kim H. and W. Wyckoff for providing a print of Fig. 2. J. B. V. was supported by National Institutes of Health Postdoctoral Fellowship GM13500. Research in B. A. A.'s laboratory is supported by National Institutes of Health Research Grant GM32117.

References 1 Walsh, C. (1979) Enzymatic Reaction Mechanisms, pp. 179-209, W. H. Freeman 2 Cullis, P. M. (1987) in Enzyme Mechanisms Page, M. I. and Williams, A., eds), pp. 178-220, Royal Society of Chemistry 3 Hendry, P. and Sargeson, A. M. (1990)in Progress in Inorganic Chemistry Vol. 38 (Lippard, S. J., ed.), pp. 201-258, John Wiley 4 Buchwald, S. J., Friedman, J. M. and Knowles,

THE STRUCTURAL and functional relationships that exist between proteins from different organisms provide insights into the evolutionary origins and possible physiological specializations of these molecules. Many proteins that perform the same function in different species (e.g. hemoglobins of vertebrate animals) are also structurally related, and may be viewed as having evolved in parallel from a common ancestral molecule. Other proteins which appear to be structurally related, yet perform different functions (e.g. lysozyme and ~-lactalbumin) may have arisen by divergent evolution from a common ancestor ]. Finally, some proteins which appear to be structurally unrelated (e.g. the mammalian and subtilisin-like serine proteases 2) but perform similar biological roles, may have arisen by convergent evolution from ancestral molecules derived from independent genetic origins. Glycolysis is considered to be one of the earliest pathways of carbohydrate H. G. Lebherz is at the Department'of Chemistry, San Diego State University,San Diego, CA 92182-0328, USA. Formerlyat the same address, ]. J. Marsh is now at the Department of Pulmonary and Critical Care Medicine, Universityof California San Diego, San Diego, CA 92103, USA.

110

J. R. (1984) J. Am. Chem. Soc. 106, 4911-4916 5 Coleman, J. E. and Gettins, P. (1983) in Metal Ions in Biology Vol. 5 (Sprio, T. G., ed.), pp. 153-217, John Wiley 6 Coleman, J. E. and Gettins, P. (1983) Adv. Enzymol. Relat. Areas Mol. Biol. 55, 381-452 7 Fernley, H. N. and Bisaz, S. (1968) Biochem. J. 107, 279-283 8 Jones, S. R., Kindman, L. A. and Knowles, J. R. (1978) Nature 275, 564-565 9 Kim, E. E. and Wyckoff, H. W. (1991) J. Mol. Biol. 218, 449-464 10 Sprio, T. G. (ed.) (1983) Metal Ions in Biology: Zinc Enzymes, Vol. 5, John Wiley 11.Hough, E. et al. (1989) Nature 238, 357-360 12 Beese, L. S. and Steitz, T. A. (1991) EMBO J. 10, 25-33 13 Kuranova, I. P. (1989) BioKhim. 53, 1567-1572 14 Buchwald, S. L., Saini, M. S., Knowles, J. R. and Van Etten, R. L. (1984) J. Biol. Chem. 259, 2200-2213 15 Van Etten, R. L. (1982)Ann. N.Y. Acad. Sci. 390, 27-51 16 Bazan, J. F., Fletterick, R. J. and Simon, J. P. (1989) Proc. Natl Acad. Sci. USA 86, 9642-9646 17 Taga, E. M. and Van Etten, R. L. (1982) Arch. Biochem. Biophys. 214, 505-515

18 Baldwao, C. E. M., Guna, E., Bittencurt, H. M. S. and Chaimovich, H. (1975) Biochim. Biophys. Acta 391, 316-325 19 Saini, M. S., Buchwald, S. L., Van Etten, R. L. and Knowles, J. R. (1981) J. Biol. Chem. 256, 10453-10455 20Zhang, Z-Y. and Van Etten, R. L. (1991) J. Biol. Chem. 266, 1516-1525 21 Doi, K., Antanaitis, B. C. and Aisen, P. (1988) Struct. Bonding 70, 1-26 22 Vincent, J. B. and Averill, B. A. (1991) FASEBJ. 4, 3009-3014 23 Vincent, J. B., Olivier-Lilley, G. L. and Averill, B. A. (1990) Chem. Rev. 90, 1447-1467 24 Vincent, J. B., Crowder, M. W. and Averill, B. A. (1991) J. Biol. Chem. 266, 17737-17740 25 Sillen, L. G. (1959) Q. Rev. Chem. Soc. 13, 146 26 David, S. S. and Que, L., Jr (1990) J. Am. Chem. Soc. 112, 6455-6463 27 Ballou, L. M. and Fischer, E. H. (1986) in The Enzymes, Vol. 17(Boyer, P. D., ed.), pp. 311-361, Academic Press 28 Vincent, J. B. and Averill, B. A. (1990) FEBS Lett. 263, 265-268 29 Martin, B. L. and Graves, D. J. (1986) J. Biol. Chem. 261, 14545-14550 30 King, M. M. and Huang, C. Y. (1984) J. Biol. Chem. 259, 8 8 4 7 4 8 5 6 31 Wo, Y-Y. P. et al. Biochemistry(in press)

Two mechanistically distinct forms of fructose-bisphosphate aldolase are known to exist. It has been assumed that the Class II (metallo) aldolases are evolutionarily more primitive than their Class I (Schiff-base) analogs since the latter had only been found in eukaryotes. With the identification of prokaryotic Class I aldolases, we present here an alternative scheme of aldolase evolution. This scheme proposes that both aldolase classes are evolutionarily ancient and rationalizes the Observed highly variable expression of both enzyme types in contemporary life forms.

metabolism to have evolved, and hence its component enzymes must be evolutionarily quite primitive. At least two of the three evolutionary processes mentioned above may have participated in establishing the current repertoire of molecular forms of the glycolytic enzyme, fructose-bisphosphate aldolase. The process of convergent evolution of unrelated proteins undoubtedly produced the two mechanistically distinct Class 1 and Class II forms of fructosebisphosphate aldolase 4, while the similar structural and functional properties

of skeletal muscle aldolases from vertebrate animals can be viewed as an example of parallel evolution3. During the 1950s and 1960s4, it was shown that fructose-bisphosphate aldolases from diverse organisms could be divided into two basic categories depending on the mechanism they use to catalyse the reversible cleavage of fructose bisphosphate to dihyroxyacetone phosphate and glyceraldehyde 3-phosphate. Class 1 aldolases (typified by the rabbit muscle enzyme) form a covalent 'Schiff-base' intermediate be-

TIBS 17 -

MARCH 1992 TaMe I. Structural and catalytic properties of representative Class I and Class II aldolases Class la

Property

Subunit size (kDa)

Archaebacteria Halobacterium vallismortis

Eubacteria Micrococcus aerogenes

Class lib Eukaryotes Rabbit muscle

Archaebacteria Eubacteria Halobacterium Escherichiacoli mediterranei

Eukaryotes Saccharomyces cerevisiae

27

33

40

NDc

39

40

Homodecamer

Monomer

Homotetramer

ND

Homodimer

Homodimer

No

No

Yes

No

Yes

Yes

Bound metal ion

None

None

None

Fe2+

Zn2+

Zn2+

Schiff-base formation

Yes

Yes

Yes

No

No

No

Carboxy-peptidasesensitivity (% residual fructose bisphosphate (FDP) cleavage act)

100

100

8

ND

100

94

Broad (7.2~8.5)

Broad (6.54.5)

Broad (6.9-8.8)

Narrow (7.2)

Narrow (7.5)

Narrow (7.0)

Native structure Full sequence available

pH optimum for FDP cleavage

aData taken from Refs 4,5,8,10,18,29. bData taken from Refs 4,13,14,21. cND, not determined.

tween the dihydroxyacetone-phosphate moiety of the substrate and an C-amino group of an active-site lysine residue during catalysis4,5. In contrast, Class II aldolases (typified by the yeast enzyme) instead utilize a divalent cation (usually Zn2+ or Fe2÷) as an electrophile in the catalytic cycle4. This striking distinction in mechanism, together with a number of other major differences in the structural and functional properties of these enzymes, led Rutter4 to propose that Class I and Class II aldolases were derived from evolutionarily unrelated ancestral molecules, and therefore, that these two aldolase types should be strictly classified as functionally analogous enzymes4. In this article, we suggest an alternative to the scheme of aldolase evolution, proposed by Rutter4 in 1964, particularly with respect to the probable evolutionary histories of the Class 1 (Schiff-base) and Class II (metallo) fructose-bisphosphate aldolases. Our scheme provides an explanation for the highly variable patterns of expression of Class I and/or Class II aldolases in related contemporary life forms and argues against previous speculations which imply that, during evolution, Class I aldolases may have become better suited to function in hexose syn-

thesis (gluconeogenesis) than their Class lI counterparts.

have

Structural relationships between Class I and Class II aldolases

The major structural and catalytic properties of some representative Class I and Class II aldolases are summarized in Table 1. As shown, each class of aldolase may be expressed in both prokaryotic and eukaryotic organisms. However, until recently, there was little information concerning the evolutionary relationships which may exist between aldolases within each enzyme family. Class I aldolases from a wide array of eukaryotic organisms have been isolated and characterized. These enzymes are invariably tetramers with subunit molecular masses of about 40 kDa, and all possess carboxy-terminal tyrosine residues that are required for maximal catalytic activity4,5. Comparisons of the amino acid sequences of aldolases from plant (maize), protozoan (trypanosome), insect (Drosophila), and mammalian (rat, human) sources suggest that the primary structures of the Class I eukaryotic enzymes have been highly conserved during evolution (Refs 6, 7 and J. J. Marsh, PhD thesis, 1990). Detailed structural information on Class I prokaryotic aldolases is limited

at present. However, some significant differences are known to exist between the prokaryotic and eukaryotic Class I aldolases in terms of their structural and catalytic properties (Table I). For example, the prokaryotic enzymes tend to show substantial variability in their subunit size and oligomeric structure, and their catalytic activities are generally found to be insensitive to carboxypeptidase treatment, suggesting that they may not possess the functional carboxy-terminal tyrosine residue which is characteristic of the eukaryotic Class I aldolases. Indeed, residues other than tyrosine have been found at the carboxyl terminus of several Class I prokaryotic aldolases ~-1°. The tryptic peptides containing the active-site lysine residue of the Micrococcus aerogenes 8 and Staphylococcus aureus 9 aldolases have been isolated and characterized. The amino acid sequence identity between the peptide from S. aureus aldolase and that derived from the prototypic Class I rabbit muscle aldolase is only about 20%, considerably less than that exhibited between the active site peptides from diverse Class I eukaryotic aldolases (greater than 70%; J. J. Marsh, PhD thesis, 1990). Even so, the distribution of hydrophobic and hydrophilic residues, as well as the predicted secondary structures of the S. aureus and

TIBS 17 - MARCH 1992

Eukaryotes

Archaebacteria

II

I

II Animals Yeast Mycobacterium smegmatis Blue-green algae Plants Fungi Staphylococci Staphylococcus caseolyticus Protozoa Peptococci Peptococci Algae Rhodopseudomonas spheroides I and II I and II Staphylococci Euglena I

Halobacterium saccharovorum Halobacterium halobium Halobacterium R- 113 Halobacterium mediterranei Halobacterium vallismortis Halobacterium volcanii Halobacterium CH-1

I and II none reported

Eubacteria II

I

Mycobacteriurn tuberculosis Lactobacillus casei Escherichia coil

Chlorella Chlamydomonas Chondrus Ochromonas

~

~

'

"

-

-

I and I17

~I / ' ' " / ~

Progenote

Rgure 1 Representative distribution of Class I and Class II fructose-bisphosphate aldolases among primary kingdoms. The division into three primary kingdoms is based on the work of Woese22. Data for archaebacteria taken from Ref. 21, for eukaryotes Refs 4,26, and for eubacteria Refs 4,12,19,20,25,26.

eukaryotic aidolase active-site peptides are very similaP, suggesting some evolutionary structural relationship in this catalytically essential segment of the protein. We have recently determined the amino acid sequence of the aminoterminal region (29 residues) of the eubacterial Class I aldolase from Mycobacterium smegmatis u, an enzyme which is similar to eukaryotic Class I aldolases in terms of its subunit molecular mass and tetrameric structure ~3. Even though this partial amino acid sequence could optimally be aligned with the amino-terminal region of diverse eukaryotic aldolases, only a 13-27% sequence identity was observed, and all of the identities involved frequently occurring amino acids such as glycine and alanine (J. J. Marsh, PhD thesis, 1990). Finally, although not as sensitive as amino acid sequence analysis, comparisons of the amino acid compositions of a number of Class I eubacterial and eukaryotic aldolases suggest that the structures of these two groups of aldolase are not closely related (Ref. 11 and J. J. Marsh, PhD thesis, 1990). Studies of prokaryotic and eukaryotic Class II aldolases have shown these enzymes to be quite similar in terms of their structural and catalytic properties (Table I, Ref. 5). Comparison of the amino acid sequences of a prokaryotic (Escherichia coil) ~4 and a e~Jkaryotic (yeast) TM Class II aldolase clearly indicates that these proteins are sequentially related and presumably arose from the same genetic background. On the other hand, sequence comparisons between

inal scheme 4. It is also now recognized that there are three, rather than two, primary evolutionary kingdoms and that a major change has occurred in the evolution of current-day organisms. In 1981, Woese 22 proposed that there are two separate prokaryotic lineages, the Evolutionary histories of Class I and Class II eubacteria, and the archaebacteria. The designation of the archaebacteria as a aldolases Based on the data available in 1964, separate kingdom is largely based on Rutter 4 proposed that Class II fructose- the fact that, in terms of molecular cribisphosphate aldolases are evolution- teria, the archaebacteria appear to be arily more primitive than the Class I as distinct from the eubacteria as they forms of this enzyme: appearance of the are from the eukaryotes=. In fact, first Class I fructose-bisphosphate recent comparative biochemical studaldolase molecule was envisioned to ies suggest that the archaebacteria may have occurred rather late during evol- actually be more closely related to ution (and only in the eukaryotic lineage eukaryotes than they are to eubacof life forms), presumably first being teria 23. The divergence of the three priexpressed in a progenitor of the current- mary kingdoms is now believed to have day unicellular green algae4. This notion occurred very early in the evolutionary was supported primarily by the phylo- process from a hypothetical common genetic distribution of aldolases ob- ancient life f~)rm:the progenote 22. In formulating our revised scheme of served at that time. Class I aldolases had only been found in eukaryotes aldolase evolution, we used accepted (algae, protozoa, plants and animals) rules of contemporary systematics to while Class II aldolases were found in describe the patterns of expression of prokaryotes (bacteria, blue--green algae) the two classes of aldolase in biological and eukaryotic yeast and fungi4; both systems. Formally, a particular phenoclasses were found in the unicellular typic expression is referred to as a green algae, Euglena and Chlamydo- character state and a given character monas 1~-17. state is considered as being either The existence of Class I fructose bis- primitive or derived, depending on the phosphate aidolases in prokaryotic diversity of present-day taxa in which it organisms was first reported in the is expressed 24. The exclusive use of early 1970s (Refs 18, 19) and sub- L-amino acids in protein synthesis is an sequent observations have demonstrated example of a primitive character state that Class I aldolases are expressed in a that has been retained throughout the wide array of prokaryotic life forms2°.2L entire spectrum of contemporary life These findings were not easily accom- forms. A derived character state, on the modated within the context of the orig- other hand, is one which is expressed Class 1 and Class lI aldolases, from any known sources, have revealed no obvious similarity between the two types 13, supporting the view that Class I and Class II aldolases arose from separate evolutionary origins.

TIBS 17 -

MARCH 1992

only in a limited number of taxa which belong to a larger group of evolutionarily related organisms. Hair, as a protective covering by mammals, could be viewed as the acquisition of a derived character, since mammals are the only subgroup of vertebrate animals that express this character state. The distribution of Class I and Class II aldolases among members of the eukaryotic, eubacterial and archaebacterial kingdoms is outlined in Fig. 1. Both Class I and Class II aldolase activities are expressed by some members of all three primary kingdoms. It follows that the capacity to express both types of aldolase was presumably available to the hyopothetical progenote. Thus, it would be impossible to classify one type of aldolase as being more primitive than the other. According to the rules of systematics, one should view the expression of one or both types of aldolase by any contemporary life form as resulting from retention of a primitive character. The fact that most contemporary organisms express only one type of aldolase activity suggests that, in most cases, expression of the other type has been lost during the course of evolution.

Function of Class I and Class II aldolases in vivo The highly variable phylogenetic expression of Class I and Class II aldolases throughout nature raises questions concerning the specific physiological roles that these two types of enzymes may play in vivo. Circumstantial evidence has led to the speculation that Class I aldolases are better able to perform hexose synthesis through the gluconeogenic pathway than are their Class II analogs. For example, some eubacteria express only Class II aldolase activity when grown on a glycolytic carbon source, but expression of Class I aldolase activity can be induced when they are grown instead on a gluconeogenic carbon source ]9,2°,2s. However, we argue that Class I and Class II aldolases are equally adept at performing hexose synthesis in vivo, based on the following observations. (1) Most microorganisms belonging to all three primary kingdoms constitutively express either Class I or Class II aldolase activities (but not both) when grown on glycolytic or gluconeogenic carbon sources (Refs 20, 21 and J. J. Marsh, PhD thesis, 1990). (2) The prokaryotic blue-green algae (cyanobacteria) utilize strictly a Class II

aldolase activity for hexose synthesis Thus, the expression of both classes of during the dark reactions of photosyn- aldolase activity in some organisms thesis 26, whereas the same function is when grown under specific metabolic served exclusively by a Class I aldolase conditions appears to reflect the retenin the chloroplast organelles of higher tion of an enzymatic redundancy which plants 27. (3) Most importantly, the uni- has been eliminated by the great majorcellular green alga Chlamydomonas ity of contemporary life forms during mundana utilizes a Class lI aldolase to evolution. perform hexose synthesis from acetate through the combined action of the gly- Acknowledgements oxylate cycle and the gluconeogenic This work was supported in part by pathway (a light-dependent process NIH grant GM-23045. We thank Ms Marie known as photoassimilation) 28, while a Ayers and Mrs LaVerna Armistead for Class I aldolase is used to synthesize their secretarial support. hexose sugars during the dark reactions of photosynthesis Is. Growth of this References organism on acetate actually decreases 1 Arnon, R, (1977) In Immunochemistry of Enzymes and their Antibodies (Salton, M. R. J., the expression of Class I aldolase activity ed.), pp. 1-28, John Wiley & Sons and increases the expression of Class II 2 Kraut, J. (1971) In The Enzymes, 3rd edn, Vol. 3 aldolase activity, which is precisely the (Boyer, P. D., ed.), pp. 547-560, Academic Press 3 FothergilPGilmore, L. A. (1987) Biochem. Soc. opposite pattern of expression observed Trans. 15, 993-995 when the same organism is grown photo4 Rutter, W. J. (1964) Fed. Proc. 23, 1248-1257 autotropically~s, despite the fact that 5 Morse, D. E. and Horecker, B. L. (1968) Adv. aldolaseis presumably functioning in a Enzymol. Relat. Areas Mol. Biol. 31, 125-181 6 Kelly, P. M. and Tolan, D. R. (1986) Plant gluconeogenic capacity in either case. Physiol. 82, 1076-1080 Taken together, these observations 7 Freemont, P. S., Dunbar, B. and Fothergillsuggest that, in vivo, Class I and Class II Gilmore, L. A. (1988) Biochem. J. 249, aldolases are performing similar cata779-788 8 Lebherz, H. G., Bradshaw, R. A. and Rutter, W. J. lytic functions. (1973) J. Biol. Chem. 248, 1660-1665 The simultaneous expression of both 9 Fischer, S. and Tsugita, A. (1982) Eur. J. types of aldolase activity by some Biochem. 128, 343-348 organisms probably represents a cata- 10 AItekar, W. and Krishnan, G. (1991) Eur. J. Biochem. 195, 343-350 lytic redundancy that has been largely 11 Marsh, J. J., Wilson, K. J. and Lebherz, H. G. eliminated by most organisms during (1989) Plant Physiol. 91, 1393-1401 evolution. Elimination of this redundancy 12 Jayanthi Bai, N. et al. (1975) Arch. Biochem. Biophys. 168, 230-234 could have occurred in a number of P. R. et al. (1989) Biochem. J. 257, ways, including (1) the constitutive 13 Alefounder, 529-534 repression of a functional structural 14 Schwelberger, H. G., Kohlwein, S. D. and Paltauf, F. (1989) Eur. J. Biochem. 180, gene which codes for one of the 301-308 aldolase types, (2) modification of one 15 Russell, G. K. and Gibbs, M. (1967) Biochim. of the two types of aldolase structural Biophys. Acta 132, 145-154 genes such that the modified gene now 16 Latzko, E. and Gibbs, M. (1969) Plant Physiol. 44, 295-300 has the status of a pseudogene or (3) outright elimination of one of the two 17 Guerrini, A. M., Cremona, T. and Preddie, E. C. (1971) Arch. Biochem. Biophys. 146, types of aldolase structural genes from 249-255 the genome altogether. Studies utilizing 18 Lebherz, H. G. and Rutter, W. J. (1973) J. Biol. Chem. 248, 1650-1659 cDNAs which code for Class I and Class 19 Stribling, D. and Perham, R. N. (1973) Biochem. II aldolases should be helpful in distinJ. 131, 833-841 guishing between the above possi- 20 Fischer, S., Luczak, H. and Schleifer, K. H. (1982) FEMS Microbiol. Lett. 15, 103-108 bilities.

Conclusions Most contemporary life forms constitutively express either Class I or Class II aldolase activities (but not both) to carry out hexose synthesis and hexose breakdown. Therefore, in vivo, the two classes of aldolase appear to perform equally well in both directions of carbohydrate metabolism. The capacity to express both types of aldolase activity was presumably available to the ancestral life forms which gave rise to extant organisms.

21 Dhar, N. M. and Altekar, W. (1986) FEMS Microbiol. Lett. 35, 177-181 22 Woese, C. R. (1981) Sci. Am. 244, 98-122 23 Iwabe, N. et al. (1989) Proc. Natl Acad. Sci. USA 86, 9355-9359 24 Uzzel, T. and Spolsky, C. (1974) Am. Sci. 62, 334-343 25 Scamuffa, M. D. and Caprioli, R. M. (1980) Biochem. Biophys. Acta 614, 583-590 26Willard, J. M. and Gibbs, M. (1968) Plant Physiol. 43, 793-798 27 Lebherz, H. G., Leadbetter, M. M. and Bradshaw, R. A. (1984) ./. Biol. Chem. 259, 1011-1017 28 Eppley, R. W., Gee, R. and Saltman, P. (1963) Physiol. Plant. 16, 777-792 29 Tolan, D. R. et al. (1984) J. Biol. Chem. 259, 1127-1131

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