Phylogenetic Analysis of ADP-GlucosePyrophosphorylase Subunits Reveals a Roleof Subunit Interfaces in the Allosteric Properties ofthe Enzyme1[C][W][OA]
Nikolaos Georgelis2, Janine R. Shaw, and L. Curtis Hannah*
Program in Plant Molecular and Cellular Biology and Horticultural Sciences, University of Florida,Gainesville, Florida 32610–0245
ADP-glucose pyrophosphorylase (AGPase) catalyzes a rate-limiting step in glycogen and starch synthesis in bacteria andplants, respectively. Plant AGPase consists of two large and two small subunits that were derived by gene duplication. AGPaselarge subunits have functionally diverged, leading to different kinetic and allosteric properties. Amino acid changes that couldaccount for these differences were identified previously by evolutionary analysis. In this study, these large subunit residueswere mapped onto a modeled structure of the maize (Zea mays) endosperm enzyme. Surprisingly, of 29 amino acids identifiedvia evolutionary considerations, 17 were located at subunit interfaces. Fourteen of the 29 amino acids were mutagenized in themaize endosperm large subunit (SHRUNKEN-2 [SH2]), and resulting variants were expressed in Escherichia coliwith the maizeendosperm small subunit (BT2). Comparisons of the amount of glycogen produced in E. coli, and the kinetic and allostericproperties of the variants with wild-type SH2/BT2, indicate that 11 variants differ from the wild type in enzyme properties orin vivo glycogen level. More interestingly, six of nine residues located at subunit interfaces exhibit altered allosteric properties.These results indicate that the interfaces between the large and small subunits are important for the allosteric properties ofAGPase, and changes at these interfaces contribute to AGPase functional specialization. Our results also demonstrate thatevolutionary analysis can greatly facilitate enzyme structure-function analyses.
ADP-glucose pyrophosphorylase (AGPase) cata-lyzes the conversion of Glc-1-P (G-1-P) and ATP toADP-Glc and pyrophosphate. This reaction representsa rate-limiting step in starch synthesis (Hannah, 2005).AGPase is an allosteric enzyme whose activity is reg-ulated by small effector molecules. In plants, AGPase isactivated by 3-phosphoglyceraldehyde (3-PGA) anddeactivated by inorganic phosphate (Pi).Plant AGPase is a heterotetramer consisting of two
identical large and two identical small subunits. Thelarge and small subunits of AGPase were generated bya gene duplication. Subsequent sequence divergence
has given rise to complementary rather than inter-changeable subunits. Indeed, both subunits areneeded for AGPase activity (Hannah and Nelson,1976, Burger et al., 2003). Biochemical studies haveindicated that both subunits are important for catalyticand allosteric properties (Hannah and Nelson, 1976;Greene et al., 1996a, 1996b; Ballicora et al., 1998;Laughlin et al., 1998; Frueauf et al., 2001; Kavakli et al.,2001a, 2001b; Cross et al., 2004, 2005; Hwang et al.,2005, 2006, 2007; Kim et al., 2007; Ventriglia et al., 2008).Surprisingly, Georgelis et al. (2007, 2008) showed that,in angiosperms, the small subunit is under greaterevolutionary pressure compared with the large sub-unit. Detailed analyses have shown that the greaterconstraint on the small subunit is due to its broadertissue expression patterns compared with the largesubunit and the fact that the small subunit mustinteract with multiple large subunits.
Large subunits have undergone more duplicationevents than have small subunits (Georgelis et al.,2008). This has led to the creation of five groups oflarge subunits that differ in their patterns of tissue ofexpression (Akihiro et al., 2005; Crevillen et al., 2005;Ohdan et al., 2005). Crevillen et al. (2003) studied thebiochemical properties of four Arabidopsis (Arabidop-sis thaliana) AGPases consisting of the four differentlarge subunits and the only functional small subunit inArabidopsis. The different AGPases had different ki-netic and allosteric properties. More specifically, the
1 This work was supported by the National Science Foundation(grant nos. IBN–0444031 and IOS–0815104 to L.C.H.) and the U.S.Department of Agriculture Competitive Grants Program (grant nos.2006–35100–17220 and 2008–35318–18649 to L.C.H.).
2 Present address: Department of Biology, Pennsylvania StateUniversity, State College, Pennsylvania 16802.
* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:L. Curtis Hannah ([email protected]).
[C] Some figures in this article are displayed in color online but inblack and white in the print edition.
[W] The online version of this article contains Web-only data.[OA] Open Access articles can be viewed online without a sub-
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AGPases differed in their affinity for the allostericregulator 3-PGA and the substrates G-1-P and ATP.This possibly reflects the different 3-PGA, G-1-P, andATP levels in the various tissues. This evidence indi-cates that not only did the different large subunitgroups subfunctionalize in terms of expression, butalso these groups may have specialized in terms ofprotein function. While the study of Crevillen et al.(2003) pointed to functional specialization of the largesubunit, the identity of the amino acid sites in the largesubunit that account for these kinetic and allostericdifferences was not pursued.
Georgelis et al. (2008) presented supporting evi-dence for AGPase large subunit specialization byidentifying positively selected amino acid sites in thephylogenetic branches following gene duplicationevents. We also identified amino acid residues thatwere conserved in one large subunit group but notconserved in another large subunit group (type Ifunctional divergence; Gu, 1999) and amino acid res-idues that are conserved within large subunit groupsbut are variable among large subunit groups (type IIfunctional divergence; Gu, 2006). Positively selectedtype I and type II sites could have contributed tospecialization of the different large subunit groups.Indeed, positively selected type II sites in severalproteins have been proven via site-directed mutagen-esis (Bishop, 2005; Norrgard et al., 2006; Cavatortaet al., 2008; Courville et al., 2008) to be important forprotein function and functional specialization. Addi-tionally, several positively selected type I and type IIamino acid sites in the large AGPase subunit identifiedin our previous evolutionary analysis (Georgelis et al.,2008) have been implicated in the kinetic and allostericproperties and heat stability of AGPase. The role ofthese sites was demonstrated by site-directed muta-genesis experiments of large subunits from Arabidop-sis, maize endosperm, and potato (Solanum tuberosum)tuber (Ballicora et al., 1998, 2005; Kavakli et al., 2001a;Jin et al., 2005; Linebarger et al., 2005; Ventriglia et al.,2008). These analyses indicate that the rest of theamino acid sites identified as positive type I and type IIsites in our previous evolutionary analysis (Georgeliset al., 2008) represent promising candidate targets formutagenesis.
To identify large subunit amino acids that are pos-sibly important in controlling enzyme properties andthat may have contributed to large subunit specializa-tion, we conducted site-directed mutagenesis of themaize endosperm large subunit encoded by Shrunken-2(Sh2). We specifically identified amino acids of SH2that correspond to amino acid sites that were detectedas positive type I and type II sites during the large sub-unit evolution (Georgelis et al., 2008). We then replacedthe SH2 residues with amino acids of a group differentfrom the SH2 family. Several amino acid sites importantfor the kinetic and allosteric properties and heat sta-bility of AGPase were identified. Our results indicatethat the subunit interfaces between the large and smallsubunits are important for the allosteric properties of
AGPase. They also indicate that amino acid changes atsubunit interfaces have been important for AGPasespecialization in terms of allosteric properties. Theseexperiments also support the idea that the majority ofpositively selected sites as detected by codon substi-tution models (Nielsen and Yang, 1998; Yang et al.,2000) and type II sites are not false positives. Site-directed mutagenesis of such sites can greatly facilitateenzyme structure-function analyses.
RESULTS
Previous Phylogenetic Analysis and Structural Mappingof Type II and Positively Selected Sites
AGPase large subunits can be placed into fivegroups depending on sequence similarity and tissuesof expression (Supplemental Fig. S1; Georgelis et al.,2007, 2008). Group 4 includes only two sequenceswhose role has not been studied. Accordingly, werestricted further evolutionary analysis to the remain-ing four groups. We identified 21 type II sites (Fig. 1;Supplemental Table S1) by doing an analysis of all ofthe pairwise comparisons between the different largesubunit groups shown in Supplemental Figure S1(Georgelis et al., 2008). These amino acid sites poten-tially contributed to the functional divergence amongAGPase large subunits. Type II sites 96 and 106 wereshown to play an important role in enzyme catalysis(Ballicora et al., 2005), while site 506 has been impli-cated in the allosteric properties of the potato tuberAGPase (Ballicora et al., 1998). We also detected 18amino acid sites upon which potential positive selec-tion may have taken place in the tree branches follow-ing the gene duplications that led to the differentlarge subunit groups (Fig. 1; Supplemental Table S1;Georgelis et al., 2008). These sites could also be im-portant in large subunit specialization, since func-tional diversification among different large subunitscould have been beneficial for the fitness of the plant.Positively selected sites 104, 230, 441, and 445 areimplicated in the allosteric properties of AGPase(Kavakli et al., 2001a; Ballicora et al., 2005; Jin et al.,2005). Finally, we identified 91 type I sites. These sitesare apparently important for AGPase function in onegroup but not in another group, and they couldcontribute to subfunctionalization or specializationor both among large subunit groups. The usefulness,however, of type I sites in detecting functional diver-gence has been disputed (Philippe et al., 2003), sincetype I divergence between orthologous and paralo-gous groups is indistinguishable in some instances(Gribaldo et al., 2003). It would be expected thatparalogues should exhibit more functional divergencecomparedwith that revealed by orthologues. Therefore,there may be a considerable number of false-positivesites of type I divergence that do not necessarily rep-resent functional divergence.
The fact that several type II and positively selectedsites have already been shown to be important for the
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kinetic and allosteric properties of AGPase stronglysuggests that the remaining type II and positivelyselected sites may also be important for enzyme func-tion. To gain insight into the potential role of thesesites, we placed them on the modeled structure of theSH2 protein. The type I sites were excluded from thisinitial analysis, since the potential inclusion of a highnumber of false positives could confound the results.Although the only plant crystal structure available
is a potato tuber small subunit homotetramer (Jinet al., 2005), the high degree of identity (40%–45%) andsimilarity (55%–65%) between the small and largesubunits strongly suggests that the structure of thephysiologically relevant AGPase heterotetramer willbe very similar or identical to the resolved homo-tetramer structure. Superimposition of SH2 and maizeendosperm small subunit (BT2) on the solved potatostructure agrees with this conjecture (Fig. 2). Addi-tionally, the potato tuber AGPase heterotetramer wasmodeled after the homotetrameric structure, and amolecular dynamics study was conducted to deter-mine the most thermodynamically favorable interac-tions between the large and small subunits (Fig. 1;Tuncel et al., 2008). Superimposition of the potatotuber large subunit on SH2 indicates that the twostructures are virtually identical (Fig. 3). This enablesus to use the potato tuber large subunit modeledstructure to determine the areas of SH2 that interactwith BT2. According to the modeled potato tuberheterotetramer (Tuncel et al., 2008), a SH2 moleculemakes direct contacts with one molecule of BT2through its C-terminal domain (tail-to-tail interaction)and to the second BT2 protein through its N-terminal
catalytic domain (head-to-head interaction), as shownin Figure 2. We observed that 17 out of 29 amino acidsites (type II and positively selected) were at or nearthe subunit interfaces (Fig. 4). The areas of SH2 thatparticipate in subunit interactions do not constitutemore than 30% of the SH2 monomer structure. Hence,almost 60% of the residues that were selected throughevolutionary analysis were located in less than 30% ofthe SH2 monomer. This preferential localization of theresidues at subunit interfaces points to the possibilitythat subunit interfaces are important for the functionalspecialization of the AGPase large subunit.
Site-Directed Mutagenesis
To determine the role, if any, of type II and positivelyselected amino acid sites, and particularly the onesfound at the subunit interfaces, in AGPase functionand to gain insight into their potential roles in largesubunit specialization, we performed site-directedmutagenesis on 12 sites in SH2. We mutagenizedseven SH2 sites (four type II, one positively selected,and two both type II and positively selected) located atthe subunit interfaces and five sites (three type II, twopositively selected) located in the rest of the SH2monomer. In all cases, the residue of SH2, whichbelongs to group 3b (Supplemental Fig. S1), waschanged to a residue found in other groups. To gainmore information about the subunit interfaces, wescanned type I sites for the ones that are located in thesubunit interfaces. We selected type I site 149 as atarget because SH2-containing group 3b contains a Hiswhile other groups contain the physicochemically
Figure 1. Amino acid alignmentbetween maize endosperm (SH2)and potato tuber large subunit. Redboxes indicate sites that make di-rect contact with the small subunitas determined by Tuncel et al.(2008). Blue and orange arrowsindicate type II and positively se-lected sites, respectively. Sites sub-jected to mutagenesis in this studyare marked with asterisks.
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different Ala or Ser. His was changed to a Ser. We alsoselected type I site 361, which is also located in subunitinterfaces. This site is invariant in group 2 but variablein group 3b. Group 3b can be subdivided in two sub-groups, one that contains only endosperm-specificlarge subunits, including SH2, and one that includesmostly embryo large subunits. SH2 along with theother members of the former subgroup contain a Thrat site 361, while the latter subgroup contains a Cys.The Thr of SH2 was changed to a Cys, which hasdifferent physicochemical properties.
Glycogen Production
Wild-type SH2 and the 14 SH2 variants created bysite-directed mutagenesis were expressed with wild-type BT2 in Escherichia coli strain AC70R1-504 (see“Materials and Methods”), and the resulting glycogenof each genotype was quantified. The majority (10 of14) of the SH2 variants resulted in altered amountsof glycogen (Fig. 5). This strongly suggests that themajority of the mutations introduced in SH2 were notneutral, at least when expressed in E. coli, despite the
fact that the substituted amino acid residues are pres-ent in other large subunit groups.
Expression of the Sh2 mutants without the presenceof the BT2 protein in E. coli resulted in no glycogenproduction (data not shown), indicating that potentialSH2 homotetramers are inactive. It is also known thatwild-type SH2 and BT2 homotetramers do not pro-duce any glycogen in E. coli (Georgelis and Hannah,2008). Hence, the changes in glycogen production ofthe Sh2 mutants are most likely due to altered prop-erties of the SH2/BT2 heterotetramer.
Characterization of Kinetic and Allosteric Properties ofSH2 Variants
Glycogen levels suggested that some of the mutantsalter AGPase function at the protein/enzyme level.Therefore, the SH2 variants and wild-type SH2 wereexpressed in E. coli along with wild-type BT2, and theresulting heterotetramers were purified (see “Mate-rials and Methods”). The affinity of the SH2/BT2complexes for the allosteric activator 3-PGA (Ka) wasdetermined in the forward direction (G-1-P + ATP /ADP-Glc + PPi). Interestingly, seven out of the 14 SH2variants had a higher Ka compared with wild-typeSH2/BT2 (Table I). The overwhelming majority ofthem (six of seven) had an amino acid change in a siteat the subunit interfaces. Two changes were in thehead-to-head interaction areas (H149S, S163F), whilefour (T361C, D368S, P372A, C382F) were in the tail-to-tail interaction areas (Fig. 4). One change (Q213H) wasin the N-terminal catalytic domain far from the sub-unit interfaces (Supplemental Fig. S2). The affinity forthe deactivator Pi (Ki) was also determined in the
Figure 2. AGPase subunit interactions. The white structures correspondto the resolved structure of potato tuber small subunit homodimers.Cyan and magenta modeled structures of the small (BT2) and large(SH2) subunits of maize endosperm, respectively, are superimposed onthe structure of potato tuber small subunit homodimers. Red circlesindicate the candidate Pi-binding sites. A, Head-to-head subunit inter-action. B, Tail-to-tail subunit interaction.
Figure 3. Superimposition of maize endosperm large subunit (SH2)modeled structure (magenta) on the potato tuber large subunit modeledstructure (white). Red areas indicate sites in the potato tuber largesubunit that are proposed to make direct contact with the small subunit(Tuncel et al., 2008).
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presence of 15 mM 3-PGA by use of Dixon plots.Higher Ka values in the variants described above wereaccompanied by lower Ki (Table I). It has been pro-posed that 3-PGA and Pi are competing for binding toAGPase and that they may even bind to the same site
(Boehlein et al., 2008). Therefore, the lower affinity for3-PGA may maximize the efficiency of Pi inhibition.
The Km values for G-1-P and ATP were determinedfor all variants at 15 mM 3-PGA. Except for a 4-foldlower affinity of BT2/S163F for ATP, all the othervariants exhibited indistinguishable and wild-type Kmvalues. Similarly, most Kcat values were similar to wild-type BT2/SH2, except for BT2/C424V (approximately150%), BT2/V227R (approximately 40%), and BT2/D368S (approximately 60%). This indicates that theallosteric changes in the variants affected the affinityfor effectors to a much greater extent than the effect onthe mechanism of activation.
The purified complex of SH2/BT2 is present in threeforms: a heterotetramer, a heterodimer, and monomersof SH2 and BT2 (Boehlein et al., 2005). The SH2 andBT2 monomers have negligible activity at the levels of3-PGA, G-1-P, and ATP used in this study (Burgeret al., 2003). Additionally, Greene and Hannah (1998a)showed that the SH2/BT2 heterodimer is inactive.Therefore, all extant evidence strongly suggests thatthe AGPase activity in this study comes from the SH2/BT2 heterotetramer.
Heat Stability
The structures of the large and small subunits arealmost identical. It has been shown that the loopconnecting the C-terminal b-helix to the N-terminalcatalytic domain in the small subunit is implicated inthe heat stability of AGPase (Boehlein et al., 2009). Thisloop makes contact with the homologous loop in thelarge subunit, suggesting that the respective loop inthe large subunit is also important for heat stability.Since nine out of 14 substitutions in SH2 were in siteslocated at the subunit interfaces, including the loopdescribed above (amino acids 362–399), the heat sta-bility of the resulting variants was determined. Thevariants and wild-type BT2/SH2 were heated for
Figure 5. Glycogen produced by SH2 wild type and variants expressedin E. coli along with BT2. Asterisks indicate significant differencescompared with wild-type BT2/SH2 at P = 0.05 (Student’s t test; n = 4).[See online article for color version of this figure.]
Figure 4. Placement of all type II and positively selected amino acidson the subunit interfaces of maize endosperm large subunit (SH2). TypeI sites 149 and 361 that were changed by site-directed mutagenesis arealso placed on the structure of SH2. SH2 modeled structure (green) wassuperimposed on potato tuber large subunit modeled structure (white).Red areas indicate sites in the potato tuber large subunit that areproposed to make direct contact with the small subunit (Tuncel et al.,2008). Type I, type II, and positively selected sites detected byGeorgelis et al. (2008) are shown in yellow. A, Areas that participatein tail-to-tail interactions. B, Areas that participate in head-to-headinteractions.
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various amounts of time at 39�C, and remaining ac-tivity was determined by assaying in the forwarddirection using 20 mM 3-PGA and saturating amountsof substrates. With the exception of BT2/S163F, whichshowed a 3-fold increase in heat stability, all of theother variants were similar to wild-type BT2/SH2(Fig. 6). These results indicate that the majority ofthe mutagenized sites at the subunit interfaces have aspecific role only on the allosteric properties of AGPase.
Correlation of Kinetic and Heat Stability Data with
Glycogen Production
In general, the amount of glycogen produced by thevariants in E. coli was consistent with the kinetic data.Six of seven allosteric variants produced loweredamounts of glycogen compared with the wild type.In the case of the exceptional BT2/S163F, the Ka wasincreased and hence decreased glycogen productionmight have been expected. This was not observed.However, the higher heat stability of BT2/S163F maycounteract the increased Ka. As a result, BT2/S163Fproduces wild-type amounts of glycogen. BT2/M172T, BT2/C114A, and BT2/E438Q had wild-typekinetic properties and heat stability. Not surprisingly,they produced wild-type amounts of glycogen. BT2/V227R and BT2/C424V had lower and higher Kcat andglycogen production compared with the wild type,respectively. BT2/V502T and BT2/A508S exhibitedwild-type kinetic properties and heat stability. How-ever, glycogen production was markedly reduced inthese mutants. Perhaps these variants have reducedsolubility and/or increased susceptibility to proteasesin E. coli, or perhaps transcription or translation isreduced in these mutants. These possibilities wouldpredict reduced amounts of SH2 and/or BT2 proteinin E. coli extracts. To investigate these possibilities,
western-blot analysis was conducted on total andsoluble protein extracts from E. coli expressing wild-type BT2/SH2, BT2/V502T, and BT2/A508S. Theamount of SH2 and BT2 in both total and solubleprotein extracts is indistinguishable from that in thewild type in these two variants (Fig. 7). Therefore, thepossible explanations discussed above for the reducedglycogen produced by BT2/V502T and BT2/A508Sshould be excluded. The underlying reason for re-duced glycogen production in these variants remainsunresolved.
Interestingly, none of the SH2 variants gave a nullphenotype in E. coli. The exact ratio of 3-PGA to Piin our E. coli system is not known. Some tentativeamounts for 3-PGA and Pi are 0.5 to 0.75 mM and 5 to10 mM, respectively, depending on the type of cells
Table I. Kinetic and allosteric properties of the wild type and SH2 mutants in a complex with BT2
All reactions were performed in triplicate. The Hill coefficient for Ka values and Km values varies from0.9 to 1.3. Asterisks indicate significant differences compared with BT2/SH2 at P = 0.05. Statisticalsignificance was estimated by an F test implemented by Prizm (GraphPad).
Sample Km G-1-P Kcat Km ATP Ka 3-PGA (15 mM 3-PGA) Ki Pi
Figure 6. Heat stability of SH2 wild type and variants in a complexwith BT2. The asterisk indicates a significant difference compared withwild-type BT2/SH2 at P = 0.05 (Student’s t test; n = 6). [See onlinearticle for color version of this figure.]
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and the growth conditions (Moses and Sharp, 1972;Ugurbil et al., 1978; Ishii et al., 2007). Since the ratio of3-PGA to Pi is low, it may be expected that our AGPasevariants have very low to almost no activity in E. coli.However, maize endosperm AGPase is known tohave some low activity even in the absence of 3-PGA(5%–10% compared with the presence of saturatingamounts of 3-PGA; Boehlein et al., 2009). Pi negates thepresence of 3-PGA and brings the AGPase activitydown to the level that is observed in the absence of3-PGA (Boehlein et al., 2008). Further reduction ofactivity requires much higher amounts of Pi (Boehleinet al., 2009). This may explain why our allostericvariants do not show a null phenotype in our E. colisystem.
DISCUSSION
Structure-function analysis of AGPase has attractedintense interest, since AGPase catalyzes a rate-limitingstep in starch synthesis. An understanding of thespecific role of amino acid sites or protein motifs canfacilitate the engineering of AGPases, leading togreater starch yield in plants. A bacterial expressionsystem has facilitated the understanding of plantAGPase function, since random mutagenesis and rapidscreening of activity in E. coli are feasible. Detailedextant analyses have identified sites important forkinetic and allosteric properties and heat stability(Greene et al., 1996a, 1996b; Greene and Hannah,1998b; Laughlin et al., 1998; Kavakli et al., 2001a,2001b; Ballicora et al., 2007; Georgelis and Hannah,2008; Hwang et al., 2008). Additionally, random mu-tagenesis of these variants has led to the identificationof intragenic suppressors of initialmutants and resultedin the identification of additional sites that areimportant for allosteric properties of AGPase (Greeneet al., 1998; Kim et al., 2007). Site-directed mutagenesishas also greatly facilitated structure-function analysisof AGPase. The resolved structure of the potato tubersmall subunit homotetramer (Jin et al., 2005) alongwith structure modeling have been used to identify
candidate sites for mutagenesis (Bejar et al., 2006;Hwang et al., 2006, 2007). Additionally, evolutionarycomparison of AGPase with other pyrophosphory-lases has identified conserved amino acid sites thathave undergone site-directed mutagenesis (Ballicoraet al., 1998, 2005; Fu et al., 1998; Frueauf et al., 2001,2003).
Herein, we have identified positively selected sitesin the large subunit of AGPase and amino acid sitesthat are conserved within large subunit groups butvariable between groups. We argue that these sitesmay have been important in functional diversificationof the large subunits and, subsequently, AGPases inplants. After placing these amino acid sites on themodeled structure of SH2, we observed that the ma-jority of them were localized at tail-to-tail or head-to-head subunit interfaces. This strongly suggests that thesubunit interfaces have been important in AGPaseisoform specialization. To gain insight into the poten-tial role of the amino acid sites, especially the oneslocated at subunit interfaces, we mutagenized 12 ofthese sites in SH2. We also mutagenized two type Isites that were located at subunit interfaces for reasonsdiscussed above. The original amino acid in SH2 wasreplaced with an amino acid found in other largesubunit groups.
Interestingly, 10 of 14 SH2 variants resulted in thesynthesis of altered amounts of glycogen in E. coli.Additionally, nine of the SH2 variants showed distinctkinetic and allosteric properties compared with wild-type BT2/SH2. These results indicate that the majorityof the amino acid changes are not neutral at theenzyme/physiological level, even though the replace-ment amino acids do exist in nature in other largesubunits. This result supports and encourages the useof phylogenetic analysis software, such as PAML (fordetection of positive selection; Yang, 1997) andDIVERGE (for detection of type I and II sites; Gu andVelden, 2002), in biochemical structure-function stud-ies. DIVERGE and PAMLwere used by Georgelis et al.(2008) to detect the sites that were biochemically testedin this study.
Surprisingly, six of nine changes in SH2 that arelocated at subunit interfaces had altered allostericproperties. Indeed, these variants showed 2.4- to 10-fold increases in 3-PGA Ka and similar decreases in thePi Ki. One substitution, S163F, also resulted in a 4-foldincrease in the ATP Km, while change D368S resultedin a slight decrease in Kcat (approximately 60% of thewild type). Overall, Kcat values were not appreciablyaltered in the allosteric variants. Therefore, most ofthe changes in these variants were quite specific for Kaand Ki. This indicates that the changes introduced bymutagenesis primarily affected the affinity for theallosteric effectors rather than the mechanism of acti-vation after effector binding. These results clearlyindicate that subunit interfaces (both head-to-headand tail-to-tail interfaces) are important determinantsof the affinity for allosteric effectors. The results alsostrongly suggest that the subunit interfaces have
Figure 7. Western blot of protein extracts from E. coli cells expressingSH2, V502T, and A508S along with BT2. [See online article for colorversion of this figure.]
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played an important role in enzyme function special-ization, particularly in diversification in terms of af-finity for the allosteric effectors, 3-PGA and Pi.
Based on the potato tuber small subunit homo-tetramer (Jin et al., 2005), one potential Pi-binding site(site 3) is formed by the head-to-head interaction area(Fig. 2). Two additional potential Pi-binding sites (sites1 and 2) are between the N-terminal catalytic andC-terminal b-helix domains, with site 1 making con-tact with the loop that participates in tail-to-tail inter-actions (Fig. 2). Hence, the fact that the subunitinterfaces are important for allosteric properties isconsistent with the structural localization of the Pi-binding sites. However, most mutagenized sites arerelatively far from the Pi-binding sites and could notdirectly bind allosteric effector molecules. This indi-cates that these sites would not have been selected formutagenesis if only the structure of AGPase wereconsidered in the experimental design.
Additional evidence pointing to the importance ofthe tail-to-tail interaction area in allosteric propertiescomes from studies with potato tuber AGPase (Kimet al., 2007). Here, an amino acid change in the loopconnecting the b-helix to the catalytic domain of thepotato tuber small subunit is important for the allo-steric properties, although the site does not makedirect contact with Pi. Overall, changes in the head-to-head and tail-to-tail interaction areas of either sub-unit are likely to affect allosteric properties, especiallyaffinity for effector molecules, either because theydirectly make contact with the effectors or they changethe orientation of amino acid residues that make directcontact with the effector molecules.
SH2 site 213 is also important in allosteric proper-ties, although it is far from the subunit interfaces of thepotential effector-binding sites (Supplemental Fig. S2).This is not the first case of a large subunit residuebeing important in allosteric properties yet far fromthe potential effector-binding sites. Site 230 was im-plicated in the allosteric properties of AGPase in astudy of the potato tuber large subunit, although it isnot close to effector sites or subunit interfaces. Site 230was initially isolated through random mutagenesis,because its alteration suppressed the phenotype of anexisting allosteric variant (Kavakli et al., 2001a). Thefinding that site 213 in SH2 is important for allostericproperties highlights the importance of phylogeneticanalysis (site 230 was also identified in our evolution-ary analysis). Phylogenetic analyses identify sites thatare not readily targeted for mutagenesis based onstructure alone. The most likely scenario is thatchanges in sites 213 and 230 indirectly affect allostericproperties by changing the structure of AGPase.
Since nine out of 15 SH2 variants were at or nearsubunit interfaces, we asked whether these alterationsaffected AGPase heat stability. Alteration in heat sta-bility has been noted with at least one mutant (Greeneand Hannah, 1998b) that affects large subunit-smallsubunit interactions. The loop connecting the N-terminaldomain to the b-helix of the small subunit participates
in tail-to-tail interactions with the large subunit and isimportant for AGPase heat stability (Boehlein et al.,2009). However, only one variant, BT2/S163F, showedaltered heat stability. Either the specific changes werenot sufficient to change heat stability or the specificsites are not important for heat stability in BT2/SH2.
Apart from altering allosteric properties, our site-directed mutagenesis specifically altered the specificactivity of BT2/V227R and BT2/C424V. Site 227 islocated in the N-terminal domain, while site 424 is inthe C-terminal b-helix. Interestingly, BT2/C424V showsa 50% greater Kcat compared with wild-type BT2/SH2.This finding is supported by the fact that BT2/C424Vproduced almost 50% more glycogen compared withBT2/SH2 when expressed in E. coli. This variant aloneor in combination with other existing variants maylead to agronomic gain when expressed in plants.
BT2/C424V is the only variant among four (424,438, 502, 508; Supplemental Fig. S2) in the C-terminalb-helix (excluding the part that participates in subunitinteractions) that exhibited an enzyme phenotype.However, this does not mean that the C-terminalb-helix is not important for enzyme function. Indeed,it makes direct contact with the effector-binding sitesand in this way influences the allosteric properties ofAGPase. Sites 441, 445, and 506 are important forallosteric properties (Ballicora et al., 1998; Kavakliet al., 2001a), and they were all detected by ourphylogenetic analysis as either type II or positivelyselected sites.
Surprisingly, BT2/V502T and BT2/A508S synthe-size markedly less glycogen in E. coli, even thoughtheir kinetic properties and heat stability are indistin-guishable from the wild type. Decreased expression,decreased solubility, or increased protease susceptibil-ity in E. coli are not likely explanations for reducedglycogen synthesis (Fig. 7). Perhaps these variantsaffect a form of regulation in E. coli that hitherto hasescaped detection.
Collectively, our results indicate that the areas of thelarge subunit that participate in tail-to-tail and head-to-head interactions with the small subunit are crucialfor the allosteric properties of AGPase. It was previ-ously shown that the allosteric properties of AGPaseare determined by the functional interaction betweenthe large and small subunits (Cross et al., 2004; Kimet al., 2007). However, this is, to our knowledge, thefirst report that indicates that the interfaces betweenthe large and small subunits are important for theallosteric properties and more specifically the affinityfor 3-PGA and Pi. Additionally, the high proportion oftype II and positively selected sites in these areasindicates that the subunit interfaces have been impor-tant in functional diversification of AGPase isoforms.These results are in accord with a biochemical studyconducted by Crevillen et al. (2003) that indicated afunctional specialization of the large subunit in termsof allosteric properties. However, this study pinpointsspecific amino acids and areas in the large subunit thataccount for specialization in allosteric properties. This
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study also indicates that the specialization has takenplace mainly in terms of the affinity for 3-PGA and Pi.Indeed, the mechanism of activation seems unaffected,since the Kcat values in the presence of activators orinhibitors of the vast majority of these mutants arevery similar to wild-type AGPase.Our mutagenesis also resulted in a change in site 424
that increased the specific activity of AGPase. Thismutation may lead to agronomic gain when expressedin plants. The allosteric sites detected can also be targetsof mutagenesis to obtain variants with low 3-PGA Kaand high Pi Ki that would also be of agronomic in-terest. Variants with low Ka and high Ki can also beachieved by isolation of intragenic suppressors of ourallosteric variants through random mutagenesis.Additionally, the results show that evolutionary
analysis can substantially benefit structure-functionstudies. The present study is among the few exampleswhere a large collection of positively selected and typeII sites initially detected by phylogenetic analysis wereverified biochemically. The fact that the majority of thechanges in these sites are not neutral should encouragebiochemists to use more evolutionary analysis to studyenzyme structure-function relationships. In this study,evolutionary analysis led to the selection of severalamino acid sites that are important for enzyme func-tion. These sites would not have been selected basedsolely on the structure of AGPase. The majority of thetype II and positively selected sites alter amounts ofglycogen synthesized and/or enzymatic properties.Type I sites can also be useful in detecting sitesimportant for function, since changes in type I sites149 and 361 altered enzyme properties. However, alarge-scale study should be conducted on type I sitesto determine the false-positive rate.Overall, this study enhanced our understanding of
the evolution and structure-function relationships inAGPase, set the stage for protein engineering that maylead to increased starch yield in crops, and providedsupport for the use of evolutionary analysis to under-stand protein function.
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