Histidine mutagenesis of Arabidopsis thaliana pyruvatedehydrogenase kinase

Alejandro Tovar-Mendez1, Jan A. Miernyk1,2 and Douglas D. Randall1

1Department of Biochemistry, University of Missouri, Columbia, MO 65211, USA; 2USDA, Agricultural Research Service,

Plant Genetics Research Unit, Columbia, MO 65211, USA

Pyruvate dehydrogenase kinase (PDK) is the primaryregulator of flux through the mitochondrial pyruvate dehy-drogenase complex (PDC). Analysis of the primary amino-acid sequences of PDK from various sources reveals thatthese enzymes include the five domains characteristic ofprokaryotic two-component His-kinases, despite the factthat PDK exclusively phosphorylates Ser residues in the E1asubunit of the PDC. This seeming contradiction might beresolved if the PDK-catalyzed reaction employed a phos-pho-His intermediate. The results from pH-stability studiesof autophosphorylated Arabidopsis thaliana PDK did not

provide any support for a phospho-His intermediate. Fur-thermore, site-directed mutagenesis of the two most likelyphosphotransfer His residues (H121 and H168) did notabolish either PDK autophosphorylation or the ability totransphosphorylate E1a. Thus, PDK is a unique type ofprotein kinase having a His-kinase-like sequence but Ser-kinase activity.

Keywords: autophosphorylation; protein kinase; pyruvatedehydrogenase complex; regulatory phosphorylation; site-directed mutagenesis.

The reaction catalyzed by the pyruvate dehydrogenasecomplex (PDC) occupies a key position in intermediarymetabolism, and is subject to multiple layers of regulation[1,2]. Reversible phosphorylation is a particularly importantcontrol mechanism for mitochondrial PDC [3,4]. Multisiteserine phosphorylation of the E1a subunit of PDC by theintrinsic pyruvate dehydrogenase kinase (PDK) inactivatesthe complex, which can then be re-activated by an intrinsicphosphopyruvate dehydrogenase phosphatase [5]. Steady-state activity of the PDC is determined by the sum of thePDK and phosphopyruvate dehydrogenase phosphatasereactions.

Sequence analysis and biochemical characterization haveshown that phosphopyruvate dehydrogenase phosphatase isa unique member of the type 2C class of phosphoproteinphosphatases [6,7]. In contrast, however, PDK, and theclosely related branched-chain a-ketoacid dehydrogenasekinase (BCKDK), remain enigmatic. Despite exclusivelyphosphorylatingSer residues in theE1a subunitsof the targetcomplexes, these kinase sequences lack motifs typicallyconserved in protein Ser/Thr-kinases [8–10]. Instead, theyinclude the cardinal motifs of protein His-kinases (PHKs)

[11]. In addition, site-directed mutagenesis of conservedamino-acid residues within the N, D, F and G boxes ofPHKs has confirmed that these motifs comprise the catalyticdomain [12–14]. These motifs are located in the C-terminalregion of PDK, as with PHKs, with the presumptive H boxdistal to the catalytic domain [15]. In addition to the sequenceresemblance to PHKs, treatment with His-directed reagentsinactivated PDK [16]. However, mutagenesis of the con-served His121 residue, a potential site of His autophos-phorylation, did not abolish PDK activity [17].

Autophosphorylation has been reported for PDKs fromseveral sources [17–19]. It has been determined that one ormore Ser residues near the N-terminus of PDK, and theclosely related BCKDK, are autophosphorylated. Surpris-ingly, however, this Ser phosphorylation seems to be stable;the phosphate is not transferred to E1a [17,19]. If, inaddition to Ser autophosphorylation, there was also Hisautophosphorylation, it would not have been detected inprevious experiments [17]. The most simple interpretation ofthe existing data is that PDK is a protein Ser-kinase (PSK)with a phospho-His catalytic intermediate. The experimentsdescribed here were designed to test this hypothesis.

Four His residues are conserved among PDK sequencesfrom various organisms; His121, His168, His231, andHis233 [numbered according to the Arabidopsis thaliana(AtPDK) sequence] [20]. His121, is� 40 residues N-terminalto the H box, His168 is within the H box, and His231 andHis233 are immediately N-terminal to the N box. It hasbeen established that His233 co-operates with Glu238 inacting as a general base catalyst in the PDK reaction [12].We report the results of pH-stability studies, which indicatethat there is not a phospho-His reaction intermediate. Inaddition, the activity of AtPDK was not abolished by theH121Q mutation, the H168Q mutation, or the H121Q/H168Q double mutation. Thus, despite the sequenceresemblance to PHKs, PDK does not have a phospho-Hisintermediate and is instead a unique type of PSK.

Correspondence to D. D. Randall, Department of Biochemistry,

Schweitzer Hall, University of Missouri, Columbia, MO 65211, USA.

Fax: + 1 573 882 5635, Tel.: + 1 573 882 4847,

E-mail: randalld@missouri.edu

Abbreviations: AtPDK, Arabidopsis thaliana pyruvate dehydrogenase

kinase; BCKDK, branched-chain a-ketoacid dehydrogenase kinase;

E1, pyruvate dehydrogenase; kd-PDC, kinase-depleted pyruvate

dehydrogenase complex; MBP, maltose-binding protein; PDC,

pyruvate dehydrogenase complex; PDK, pyruvate dehydrogenase

kinase; PHK, protein histidine-kinase; PSK, protein serine-kinase.

Note: a web page is available at http://www.biochem.missouri.edu/

(Received 6 February 2002, revised 4 April 2002,

accepted 15 April 2002)

Eur. J. Biochem. 269, 2601–2606 (2002) � FEBS 2002 doi:10.1046/j.1432-1033.2002.02933.x

M A T E R I A L S A N D M E T H O D S

Reagents

Restriction endonucleases were from New England Biolabs(Beverly, MA, USA). PCR primers were from IntegratedDNA Technology, Inc. (Coralville, IA, USA). Unlessotherwise noted, reagents were provided by the SigmaChemical Company (St Louis, MO, USA).

In silico analyses

Amino-acid sequences were retrieved from the NationalCenter for Biotechnology Information web page (http://www.ncbi.nlm.nih.gov/). Sequences were initially alignedusing PHYLIP [21], and the alignments polished manually. Forphylogenetic comparisons, the AtPDK amino-acid sequencewas used as the query for a BLAST [22] search of the NCBIdatabase. The resulting sequences were aligned with PHYLIP,then subjected to 500 rounds of bootstrap analysis using theSEQBOOT function of DNAMAN (version 2.71; LynnonBioSoft, Vaudreui, Quebec, Canada). The phylogenetic treewas constructed using TREEVIEW [23]. Secondary-structure-based phylogenetic comparisons were conducted using PSI-BLAST [24] coupled to GenTHREADER [25].

Bacterial expression and purification of maltose-bindingprotein (MBP)–AtPDK

MBP–AtPDK was expressed and purified as describedpreviously [17]. After affinity purification, the protein wasconcentrated using an ultrafiltration membrane with a10-kDa cut-off (Amicon, Beverly, MA, USA), then storedin aliquots at )20 �C.

Site-directed mutagenesis

The AtPDK reading frame, previously cloned into pMal-cRI [17], was used as the template for mutagenesis. TheBamHI–PstI fragment containing the sequence that encodesthe C-terminal half of AtPDK (G160 to P366) was PCR-amplified using primers DDR229 (5¢-ctcgagctgcagctattaTCGGGTAAAGGCTCTTGCGA-3¢) and DDR 309(5¢-CGAATCGGGATCCGGATGCTTATTGGGCAGCAAGTTGAGTTGCAT-3¢). The DDR309 primer includesthe H168Q mutation (the base changed is in bold). Thermalcycling was conducted using the ACCUZYME DNApolymerase (Midwest Scientific, St Louis, MO, USA). ThePCR product was inserted into pGEM-3Zf(+) andsequenced to verify the H168Q mutation and that noadditional changes had been introduced. The H168QC-terminus fragment was then transferred to pBluescript IISK+. The N-terminus (XbaI–BamHI fragment) of eitherthe wild-type (wt) or the H121Q AtPDK sequence was thenligated to the pBluescript H168Q C-terminus, generatingan ORF encoding the H168Q or H121/168Q AtPDKmutants. These constructs were transferred to pMal-cRI forexpression.

Phosphorylation/inactivation of the PDC

Activity of the PDC was measured spectrophotometricallyas the increase in A340 due to reduction of NAD+ [26]. As a

substrate for recombinant PDK, kinase-depleted (kd)-PDCwas prepared from purified pea seedling mitochondria asdescribed previously [27]. For phosphorylation assays,MBP–AtPDK was incubated with kd-PDC for 5 min at25 �C, then reaction was initiated by adding [c-32P]ATP(Perkin Elmer Life Sciences; 2.2 TBqÆmmol)1). Reactionswere stopped by adding an equal volume of 2 · samplebuffer [8 M urea, 4% (w/v) SDS, 4% (v/v) 2-mercaptoeth-anol, 10 mM EDTA] followed by heating at 70 �C for20 min. Proteins were resolved by SDS/PAGE, transferredto nitrocellulose membranes, then examined by autoradio-graphy. Incorporation of 32P into proteins was quantified byliquid-scintillation spectrometry of excised bands.

ATPase activity was assayed essentially as described in[12], using [c-32P]ATP as the substrate.The 32Pi released fromATP was quantified by liquid-scintillation spectrometry.

Determination of the pH stabilityof autophosphorylated AtPDK

To investigate which amino-acid residues were involved,1.4 lg of either wild-type MBP–PDK or the H121Q/H168Q mutant protein was autophosphorylated with[c-32P]ATP, resolved by SDS/PAGE, transferred topoly(vinylidene difluoride) membranes (Millipore Corp.).The membranes were treated with 50 mM KCl/HCl, pH 1,100 mM Tris/HCl, pH 7, or 1 M NaOH, pH 14, in thepresence of 10% methanol for 2 h at 45 �C. Radioactivitywas then quantified by liquid-scintillation spectrometry ofthe corresponding excised bands. In parallel with AtPDK,Escherichia coli CheA was autophosphorylated for use as abona fide PHK control.

R E S U L T S

Alignment of the AtPDK sequence with those of orthologsfrom maize, mouse, and Ascaris suum reveals substantialprimary sequence conservation (Fig. 1). Sequence identity isnotably high within the H, N, D, and G boxes, whichcomprise the cardinal motifs of PHKs (Fig. 1). When theAtPDK sequence was used as the search term in BLAST

analysis of GenBank, the only sequences retrieved that hadsignificant E-values (<0.026) were those of other PDKs, theclosely related BCKDKs, and several signal-transducingPHKs. Significance was determined using the student’s2-tailed t test. The phylogenetic relationships among thesesequences are presented in Fig. 2. Similar results wereobtained when predicted secondary structures were used forcomparisons (data not presented).

From the results of previous analyses, it was concludedthat both native and recombinant PDK autophosphory-late Ser residues. The methods used, however, would nothave specifically detected the occurrence of phospho-His.To re-evaluate this question, AtPDK was autophosphor-ylated using [c-32P]ATP, then subjected to acidic (pH 1),neutral (pH 7), or alkaline (pH 14) treatment beforequantitative analysis. The E. coli CheA protein, a PHKinvolved in chemotaxis, was included as a control. The 32Plabeling of AtPDK was acid-stabile but alkali-labile(Fig. 3). The extent of 32P incorporation into the AtPDKH121Q/H168Q mutant protein was much lower than withthe wild-type protein; however, the pattern of pH stabilitywas identical. Under the same experimental conditions,

2602 A. Tovar-Mendez et al. (Eur. J. Biochem. 269) � FEBS 2002

labeling of CheA was, as expected, alkali-stable andacid-labile.

Recombinant AtPDK was capable of both time-depend-ent inactivation of kd-PDC (Fig. 4) and incorporation ofradioactivity from [c-32P]ATP into E1a (Fig. 5).The kd-PDC used as the substrate for PDK had virtuallyno associated kinase activity. Residual PDC activity after a60-min incubation in vitro was essentially identical with orwithout MgATP (Fig. 4). The H121Q/H168Q mutant ofMBP–AtPDK inactivated kd-PDC less rapidly than didwild-type MBP–AtPDK (Fig. 4), although, if assays wereextended, the same end-point was ultimately reached (datanot presented). Whereas the MBP–AtPDK chimera had fullcatalytic capability, removal of the fusion partner increasedthe rate of PDC inactivation approximately fourfold. Thelower rate of the mutant kinase was more pronounced whentested as the MBP fusion.

Mutagenesis of conserved residues His121 and His168differentially affected autophosphorylation and trans-phosphorylation of E1a (Fig. 5). The AtPDK H121Qmutant displayed an � 80% reduction in the extent ofautophosphorylation after 5 min, but had only a smalleffect on transphosophorylation of E1a. Similar results wereobtained in experiments of longer duration. The H168Q

mutant had less inhibition of autophosphorylation butmore of transphosphorylation (Fig. 5). Inhibition of bothautophosphorylation and transphosphorylation by theH121/168Q double mutant was approximately additive.

In contrast with a previous report of low-level ATPaseactivity of rat PDK2 [12], we were unable to detect anysignificant hydrolysis of ATP to ADP plus Pi by AtPDK(data not shown). The recombinant enzyme preparationsused for ATPase assays were, however, fully capable ofinactivating kd-PDC. It is not clear if the previouslyreported ATPase activity was the result of a minorcontaminant, or if this result indicates an actual differencebetween the rat and A. thaliana enzymes. This possibilityseems unlikely, considering the extent of protein sequenceconservation.

D I S C U S S I O N

A prominent regulatory mechanism for the mitochondrialPDC is multisite serine phosphorylation [2–5]; thus, PDK isa PSK. The domains that define PSK enzymes include 12conserved subdomains which fold into a common catalyticcore structure [8]. After cloning and sequence analysis,however, it was clear that mammalian PDK lacks the

AtPDK MAVKKACEMFPKSLIEDVHKWGCMKQTGVSLRYM--MEFGSKPTERNLLISAQFLHKELPZmPDK2 MASEPVARAVAEEVARWGAMRQTGVSLRYM--MEFGARPTERTLLLAAQFLHKELPMmPDK2 MRWVRALLKNASLAGAPKYIEHFSKFSPSPLSMKQFLDFGSSNACEKTSF--TFLRQELPAsPDK MFLTRRLLGPFTSAIARKLEHYSQFQPSSLTIQQYLDFGQTGTMKSSFL---FLKNELLconsensus L + ++FG+ T + +L FL++ELP

IRVARRAIELQTLPYGLSDKPAVLKVRDWYLESFRDMRAF------PEIKDSGDEKDFTQ IRIARRALDLDSLPFGLSTKPAILKVKDWYVESFREIRSF------PEVRNQKDELAFTQ VRLANIMKEINLLPDRVLGTPSVQLVQSWYVQSLLDIMEF--LDKDPEDHRTLSQFTDAL VRLANIMQEISLLPPTLLKMPSRRLVSNWYCESFEDLLQFEHAQVEPDIMSKFNDQLQTI +R+A+ E+ LP +L P++ V WY+ESF D+ F PE+ + T

MIKAVKVRHNNVVPMMALGVNQLKK--GMN---SGNLDEIHQ-FLDRFYLSRIGIRMLIG MIKMIRVRHTNVVPAIALGVQQLKKDLGGPKAFPPGIHEIHQ-FLDRFYMSRIGIRMLIG VT--IRNRHNDVVPTMAQGVLEYKDTYGDD---PVSNQNI-QYFLDRFYLSRISIRMLIN L—----KRHSRVVETMAEGLIELRESEGVD---IASERGI-QYFLDRFYINRISIRMLQN + ++ RH +VVP+MA GV LK G + + I Q FLDRFY+SRI+IRMLI ______

QHVELHNP--NP--PLHTVGYIHTKMSPMEVARNASEDARSICFREYGSAPEINIYGDPS QHVALHDPD--P-EPGVI-GLINTKMSPMTVARIASEDARAICMREYGSSPDVDIYGDPG QHTLIFDGSTNPAHPKHI-GSIDPNCSVSDVVKDAYDMAKLLCDKYYMASPDLEIQ-EVN QHLVVF-GVVLPESPRHI-GCIDPGCDVESVVHDAYENARFLCERYYLTAPGMKL--EMH QH+ + P P HI G I + S V + A E AR +C R Y ++P++ I + H-box _

----FTFP–-YV--PTHL-HLMMYELVKNSLRAVQERFVDSDRVAPPIRIIVADGIEDVT -------FTFPYVTP-HL-HLMIFELVKNSLRAVQERYMDSDKLAPPVRIIVADGAEDVT -ATNANQPIHMVYVPSHLYHML-FELFKNAMRATVESHESSLTL-PPIKIMVALGEEDLS NSVNPGMPISIVAVPSHLYHIM-FELFKNSMRATVENHGADEDL-PPIKVMVVRGAEDLS P +V P+HL H+M FEL+KNS+RA E S+ L PPI+I+VA GAED++ N-box D-box-

IKVSDEGGGIARSGLPRIFTYLYSTARNPLEEDVDLGIADVPVTMAGYGYGLPISRLYAR IKISDEGGGIPRSGLSRIFTYLYSTAENPPD---LDGHNEG-VTMAGYGYGIPISRLYAR IKMSDRGGGVPLRRIERLFSYMYSTAPTPQPGTGG-------TPLAGFGYGLPISRLYAK IKISDRGGGVSRTILDRLFTYMYSTAPPPPRDGTQPP-------LAGYGYGLPLSRLYAR IK+SD GGG+ R+ L RIFTY+YSTA P +AGYGYGLPISRLYAR G1-box G2-box

YFGGDLQIISMEGYGTDAYLHL-SRLGDSQEPLP YFGGDLQIISMEGYGTDAYLHL-SRLGDSEEPLP YFQGDLQLFSMEGFGTDAVIYLKALSTDSVERLPVYNKSAWRHHYQTIQEAGDWCVPSTE YFHGDMYLVSMEGYGTDAMIFLKAIPVEASEVLPIYSTSSRRQLTMSPQAADWSHQLPNH YF GDLQ++SMEGYGTDA++ L + DS E LP

PKNTSTYRVS GNRNL

Fig. 1. Comparison of selected PDK sequences.

At, A. thaliana PDK (GI:4049631); Zm,

Zea mays PDK2 (GI:3695005); Mm, Mus

musculus PDK2; As, Ascaris suum PKD

(GI:1945392). The sequences were aligned

using PHYLIP [21], then the alignments were

optimized manually. The conserved His

residues are shown in bold. The locations of

sequences corresponding to the five cardinal

motifs of PHKs [11,15] are indicated by

underlines. When at least three residues belong

to the same family, the consensus is indicated

as (+).

� FEBS 2002 A. thaliana pyruvate dehydrogenase kinase (Eur. J. Biochem. 269) 2603

defining domain organization of PSKs [9]. Instead PDKsequences include the five canonical domains (H, N, D, G1,and G1 boxes) of PHKs [11,15]. It was subsequently foundthat this same organization is shared by mammalian,nematode [28], fly [29], and plant (Figs 1 and 2) [10] PDKsequences, as well as those of the related BCKDKs [19].

Thus, PDK is a conundrum. The results from primarysequence analysis and at least some biochemical experi-ments [16] are consistent with PDK as a PHK. At the sametime, it has been unequivocally established that PDKphosphorylates multiple Ser residues in the E1a regulatorytarget. The simplest resolution of these apparent contradic-tions would be the occurrence of phospho-His as a reactionintermediate. This, however, does not seem to be the case.

Previous analyses employed pH conditions that wouldnot have allowed detection of the transient occurrence ofphospho-His. We directly addressed this by incubating

AtPDK with [c-32P]ATP and then determining the stabilityof the resultant phosphoenzyme. Recombinant CheA wasincluded in these assays as a PHK control. From previousresults [17] we expected that most of the radiolabel inAtPDK would be acid-stable phospho-Ser. This was thecase, and we observed no significant alkali-stable labeling of

pH1

Radio

activity (

cpm

x 1

03)

0

3

6

9

12

15

CheA wild-type H121Q/ H168Q

MBP-AtPDK

pH7

pH14

Fig. 3. Effect of pH on the stability of AtPDK autophosphorylated using

[c-32P]ATP. Approximately 1.4 lg wild-type or the H121Q/H168Q

mutant of MBP–AtPDK, or E. coli CheA, was incubated with 2 pmol

Mg[c-32P]ATP (0.22 TBqÆpmol)1) for 1 h at 25 �C. After SDS/PAGE,

the proteins were transferred to poly(vinylidene difluoride) mem-

branes, which were then treated with 50 mM KCl/HCl (pH 1), 100 mM

Tris/HCl (pH 7), or 1 M NaOH (pH 14) in the presence of 10% (v/v)

methanol for 2 h at 45 �C. The radiolabeled proteins were detected by

autoradiography, and the bands excised and quantified by liquid-

scintillation spectrometry.

Rela

tive P

DC

activity

0

0.2

0.4

0.6

0.8

1.0

0

Time (min)

10 20 30 40 50 60

Fig. 4. Inactivation of kd-PDC by AtPDK. All assay mixtures con-

tained 45 lg kd-PDC and 20 lM MgATP. Assays contained 53 lg

MBP–AtPDK (wt and mutant) or 13 lg AtPDK (wt or mutant). At

the indicated time points, samples were taken for spectrophotometric

assay of PDC activity. (n) + ATP; (m) – ATP + MBP–AtPDK; (j)

+ ATP + MBP–AtPDK; (d) + ATP + MBP–AtPDK H121Q/

H168Q; (h) + ATP + AtPDK; (s) + ATP + AtPDK H121Q/

H168Q. Data are representative results.

0.1

Sp-PDK

Mm-BCKDK

Nc-BCKDK

Ec-SHK

Ss-SHK

Am-SHK

Sc-SHK

Bh-SHK

St-SHK

At-PDK

Zm-PDK2

As-PDK

Dm-PDK

Mm-PDK

Fig. 2. Phylogenetic relationships among PDKs, BCKDKs, and sensor

histidine kinases (SHK). Sp, Schizosaccharomyces pombe (GI:7708590);

Mm, M. musculus BCKDK (GI:6753164); Nc, Neurospora crassa

(GI:12718471); Ec, E. coli BaeS (GI:2507376); Ss, Synechocystis sp.

(strain PCC 6803) SHK (GI:7469378); Am, Amycolatopsis mediterra-

nei kA SHK (GI:7339510); Sc, Streptomyces coelicolor SHK

(GI:7799270); Bh, Bacillus halodurans ResE (GI:15614144); St, Strep-

tococcus thermophilus Hpk2 (GI:13324643); At, A. thaliana PDK

(GI:4049631); Zm, Z. mays PDK2 (GI:3695005); As, A. suum PDK

(GI:1945392); Dm, Drosophila melanogaster PDK (GI:7303893); Mm,

M. musculus PDK2 (GI:8096763). The sequences were aligned using

PHYLIP, then analyzed with SEQBOOT (500 rounds of bootstrapping).

The scale bar corresponds to the number of substitutions per site. The

tree was constructed using TreeView.

2604 A. Tovar-Mendez et al. (Eur. J. Biochem. 269) � FEBS 2002

AtPDK, although this was seen with the CheA control. Themethod used for these analyses does not yield an all ornothing result, so it is not possible to unequivocally statethat there is not a phospho-His intermediate, although theresults do not support this possibility.

The phospho-His contingency was further addressedusing site-directed mutagenesis. Four His residues areconserved among PDK sequences; His121, His168,His231, and His233 (numbered according to the A. thalianaPDK sequence [20]). It has been established that His233co-operates with Glu238 in acting as a general base catalystin the PDK reaction [12], so we discounted His231 or His233as sites for His autophosphorylation. The remaining possi-bilities are His121 and His168, which are upstream of andwithin the H box, respectively. If one of these His residueswere a site of autophosphorylation, it would be expectedthat mutagenesis would inactivate AtPDK. Although theH121Q, H168Q, and H121Q/H168Q mutant proteins hadreduced PDK activity, they were each capable of bothautophosphorylation and transphosphorylation of E1a.

At this point, the function of Ser autophosphorylation inPDK activity is unclear, although it has been reported withPDKs from several sources [17–19]. Ser autophosphoryla-tion is apparently stable, and the phosphate group is notsubsequently transferred to E1a [17,19]. The His mutagen-esis of AtPDK had distinct effects on autophosphorylationand transphosphorylation of E1a. It seemed possible thatthe apparently different effects were an artefact arising fromdifferential phosphorylation of the recombinant proteins

during bacterial expression. This possibility was tested byadding 32Pi to bacterial cultures before induction of AtPDKsynthesis. However, none of the resulting AtPDK proteins,wild-type or any of the His mutants, showed any 32Plabeling. Thus the role of Ser autophosphorylation remainsenigmatic, although the results with the His mutant AtPDKproteins further support dissociation of autophosphoryla-tion from transphosphorylation of PDC.

While this manuscript was in preparation, the structure ofADP-ligated rat PDK2 was solved at 2.5 A resolution [30].The crystallographic data support earlier sequence-basedstructural predictions that PDK is a member of the PHK/ATPase superfamily [14]. The authors conclude thatHis115 (His121 in AtPDK) is not solvent accessible, andis involved in important structural hydrogen bonds withinthe N-terminal domain [30]. Thus, despite the sequencesimilarity to PHKs, results from both structural analysesand use of site-directed mutagenesis argue against any rolefor His phosphorylation in the PDK reaction.

A C K N O W L E D G E M E N T S

G. L. Hazelbauer generously provided recombinant E. coli CheA. This

research was supported by National Science Foundation grant IBN-

9876680, the Missouri Agricultural Experiment Station, and the Food

for 21st Century Program.

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kDa97.666

45

29

MBP-AtPDK

E1α

H121Q H168QH121Q/H168Qwt

A

B

Re

lati

ve

in

co

rpo

rati

on

0

0.2

0.4

0.6

0.8

1.0

wt H121Q H168Q H121Q/H168Q

E1α

MBP-AtPDK

Fig. 5. Autophosphorylation of MBP–AtPDK and transphosphoryla-

tion of PDC E1a. (A) 3 lg kd-PDC was incubated with 1 lg MBP–

AtPDK plus 200 lM Mg[c-32P]ATP (18 GBqÆmmol)1) for 5 min at

25 �C. A set of representative autoradiographs are presented. (B)

Relative incorporation of 32P into MBP–AtPDK or E1a. Data are

means ± SD from three independent enzyme preparations. The same

patterns of results were obtained using either 1.5 or 6 lg of MBP–

AtPDK.

� FEBS 2002 A. thaliana pyruvate dehydrogenase kinase (Eur. J. Biochem. 269) 2605

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