Structure, Vol. 10, 225–235, February, 2002, 2002 Elsevier Science Ltd. All rights reserved. PII S0969-2126(02)00708-6

Crystal Structure of 4-Amino-5-Hydroxymethyl-2-Methylpyrimidine Phosphate Kinase from Salmonellatyphimurium at 2.3 A Resolution

ethylthiazole kinase (THZ kinase). THZ kinase is en-coded by the thiM gene of E. coli but, unlike HMPPkinase, which is an essential thiamin biosynthetic en-zyme, THZ kinase is a salvage enzyme. The structureof THZ kinase [4] showed that this enzyme belongs to

Gong Cheng, Eric M. Bennett, Tadhg P. Begley,and Steven E. Ealick1

Department of Chemistry and Chemical BiologyCornell UniversityIthaca, New York 14853

the ribokinase family [5]. Consequently, it was expectedthat HMPP kinase would also belong to the ribokinasefamily.Summary

Members of the ribokinase family have a commoncore structure consisting of a central eight-stranded �The crystal structures of Salmonella typhimuriumsheet that is flanked by eight structurally conserved �4-amino-5-hydroxymethyl-2-methylpyrimidine phos-helices, five on one side and three on the other. Thephate kinase (HMPP kinase) and its complex with sub-active site is located in a groove that stretches out along

strate HMP have been determined. HMPP kinase cata-one edge of the � sheet, with ATP binding at one end and

lyzes two separate ATP-dependent phosphorylationthe small molecule substrate at the other; the acceptor

reactions and is an essential enzyme in the thiamin hydroxyl group of the small molecule lies near thebiosynthetic pathway. HMPP kinase is a homodimer

�-phosphate of ATP at the center of the groove. Evenwith one active site per monomer and is structurally though the structures of the monomers are similar, thehomologous to members of the ribokinase family. A family shows considerable variation in quaternary struc-comparison of the structure of HMPP kinase with other ture. Adenosine kinases from both humans [6] and Tox-members of the ribokinase family suggests an evolu- oplasma gondii [7] are monomers, ribokinase from E.tionary progression. Modeling studies suggest that coli is a dimer, in which the monomers are joined throughHMPP kinase catalyzes both of its phosphorylation a � sheet flap that covers the active site [5], and THZreactions using inline displacement mechanisms. We kinase from Bacillus subtilis is a trimer, in which thepropose that the active site accommodates the two active site forms between adjacent monomers [4]. ADP-separate reactions by providing two different binding dependent glucokinase is also a ribokinase family mem-modes for the phosphate group of HMP phosphate. ber and is monomeric [8].

Interest in HMPP kinase arises from several consider-ations. First, HMPP kinase is an essential enzyme inIntroductionthe biosynthesis of thiamin. Second, members of theribokinase family accept a wide range of substrates,The thiamin phosphate precursors 4-methyl-5-�-hydroxy-including several carbohydrates and aromatic smallethylthiazole phosphate (THZ-P) and 4-amino-5-hydro-molecules, such as THZ and HMP, all of which are phos-xymethyl-2-methylpyrimidine pyrophosphate (HMP-PP)phorylated at a hydroxymethyl group. Because HMPPare formed in separate branches of the biosynthetickinase was predicted to be a member of the ribokinasepathway and coupled by the enzyme thiamin phosphatefamily, we hoped the structure would shed light on howsynthase [1]. Thiamin phosphate is then phosphorylatedthe family members evolved to accept different smallin a final step to form thiamin pyrophosphate, the activemolecule substrates. Finally, HMPP kinase displays anform of vitamin B1. 4-Amino-5-hydroxymethyl-2-methyl-unusual dual functionality. Like other members of thepyrimidine phosphate kinase (HMPP kinase) catalyzesfamily, HMPP kinase catalyzes the phosphorylation oftwo related reactions in consecutive steps of the thiamina hydroxymethyl group. However, HMPP kinase alsophosphate biosynthetic pathway. In the first reaction,catalyzes a second type of phosphorylation, in which theHMPP kinase transfers the terminal phosphate of ATPphosphate group is transferred to the phosphomethylto the hydroxyl group of HMP to form 4-amino-5-hydro-group of HMP-P. In this paper, we report the structurexymethyl-2-methylpyrimidine phosphate (HMP-P). Thisof HMPP kinase and compare its structure to other mem-reaction is used to salvage HMP from the growth me-bers of the ribokinase family. This comparison suggestsdium. In the second reaction, HMPP kinase transfersthat the ATP binding site is conserved and that the activethe terminal phosphate from another molecule of ATPsite flap may be a morphological marker for evolutionto the phosphomethyl group of HMP-P to form HMP-PP,within the family. The structure of HMPP kinase alsothe substrate for thiamin phosphate synthase (Figure 1).offers insight into the structural basis for its bifunctional

This reaction is an essential step for the biosynthesisactivity.

of thiamin pyrophosphate.HMPP kinase is encoded by the thiD gene in Salmo-

Resultsnella typhimurium [2] and other microorganisms. Thehomologous enzyme encoded by the thiD gene of Esch- Quality of the Final Modelerichia coli is reported to function as a tetramer [3]. The structure of HMPP kinase was determined at 2.3 AAmino acid sequence alignments indicated that HMPP resolution, and the structure of the binary complex withkinase might be homologous to 4-methyl-5-�-hydroxy-

Key words: ThiD; thiamin biosynthesis; catalytic mechanism; riboki-nase family; kinase; phosphorylation1Correspondence: see3@cornell.edu

Structure226

coli HMPP kinase to be a homotetramer [3]. No evidenceis seen in the crystal structure for further association ofthe dimers into tetramers. Each monomer contains eight� helices, two 310 helices, and ten � strands with an �-�-�three-layer sandwich fold. A structure of the HMPP ki-nase monomer is illustrated in Figure 2. Each subunitcontains a central eight-stranded � sheet with topology�2�1�5�6�7�8�9�10. The strands of the central � sheetare parallel, except for strand �9, which is antiparallelto its neighboring strands. Five � helices (�2, �3, �4,�6, and �7) flank one side of the sheet, and the remainingthree � helices (�1, �8, �9) are on the opposite side. All

Figure 1. The Two Phosphorylation Reactions that are Catalyzed the � helices except �9 are approximately antiparallelby HMPP Kinase

to the strands of the central � sheet. There are twoadditional � strands (�3 and �4) joined by a short loop

substrate HMP was determined at 2.6 A resolution. Each that runs from residue 46 to 50. These two strands to-final model includes two monomers designated A and gether with loop 109–116 form a flap that folds backB, which are related by 2-fold noncrystallographic sym- over the HMP substrate, shielding it from the solvent.metry. The crystallographic R factors for unliganded The overall structure of the HMPP kinase monomer isHMPP kinase and the substrate complex are 23.5% similar to that of ribokinase [5].(Rfree � 27.5%) and 23.0% (Rfree � 27.0%), respectively.The unliganded HMPP kinase model includes 496 ofthe 532 amino acids, 4 sulfate anions, and 139 water Dimer Interactions

The active form of HMPP kinase is an elongated dimermolecules. Residues 110–115, 178–186, and 199–202are disordered and invisible in the electron density of with approximate dimension of 46 A � 85 A � 62 A

(Figure 3). The subunit interface in the HMPP kinasethe unliganded HMPP kinase. The substrate complexmodel includes 508 residues, 4 sulfate anions, 2 HMP dimer is composed of two regions. The first region is

formed by the first 70 N-terminal amino acids, consistingmolecules, and 75 water molecules. In the electron den-sity for the complex, residues 110–115 were visible, but of �1, �2, �1, �2, �3, and �4. The C-terminal loop (249–

266) forms the second part of the plane. Together, thesethe side chains were not well defined, while residues178–186 and 199–202 remained completely disordered. two regions form a relatively flat surface, which contacts

the equivalent surface in the second monomer after aIn both models, the poly-His tails at the N terminus werenot visible. 2-fold rotation. Each monomer contributes 4395 A2 of

buried surface area in the formation of the dimer. Exten-sive hydrogen bonding and hydrophobic interactionsOverall Structure

HMPP kinase is a homodimer in the crystal structure. were observed in the interface. These include 38 hydro-gen bonds between the residues from separate mono-This is in contrast to an earlier study that reported E.

Figure 2. The Structure of the HMPP KinaseMonomer

(A) Stereoview of a C� trace colored by resi-due number and with every tenth residue la-beled with a sequence number. Breaks in thebackbone are connected with dashed lines.(B) Topology diagram. The � helices areshown as rectangles, and the � strands areshown as arrows. Helices 5 and 10 are 310

helices. The dotted line depicts the disor-dered loops. Each secondary structural ele-ment is labeled in its center with its designa-tor and with its beginning and endingsequence number. The elements common toall members of the ribokinase family are inlight blue for � helices and in light green for� strands.(C) A ribbon diagram showing the overall fold(� helices, blue; � strands, green). The figurewas prepared with MolScript [35], BobScript[35–37], and Raster3D [38, 39].

Structure of HMPP Kinase227

maps, while in the HMPP kinase-HMP complex struc-ture, the loop is ordered but displays high B factors.The active site forms a pocket that buries the substratecompletely (Figure 4). The side chains of Ala18, Val42,Met80, and Val107 provide hydrophobic interactions,while Glu44 forms a hydrogen bond with the HMP4-amino group. A well-ordered water molecule is posi-tioned by hydrogen bonds to backbone atoms of Gly11and Met80. This water molecule in turn hydrogen bondsto the HMP N1 atom. A second active site water mole-cule is positioned near the first water molecule and theside chains of Asp23 and Cys213.

Figure 3. Ribbon Diagram of HMPP Kinase Dimer Viewed along the In addition to HMP, two well-ordered sulfate anions2-Fold Axis were observed in the HMPP kinase-HMP complex, re-The � helices are blue, and the � strands are green. The HMP sulting from high sulfate concentrations in the crystalli-molecules and sulfate ions are shown as ball and stick models. The zation conditions. Sulfate 1 is located within hydrogenHMP and sulfate binding sites and the binding site flap are labeled

bonding distance of Asp105, Lys176, and the hydroxylfor each monomer. The closest contact between the two HMP mole-group of HMP. Other nearby residues include Thr106,cules is about 20 A.Glu142, and Gly212. Sulfate 1 is also present in theunliganded HMPP kinase structure. Sulfate 2 is located

mers. In addition, ten water molecules (five unique and within hydrogen bonding distances of the Gly210 amidefive related by symmetry) were observed in the dimer nitrogen atom and the hydroxyl group of HMP. Gly210interface. These water molecules bridge Ser15A and is part of the anion hole that is predicted in all membersTyr33B, Asn5A and Glu251B, Arg3A and Glu251B, of the ribokinase family [5]. Sulfate 2 is not observedLys25A and Gly34B, Cys47A and Ser69B, and their sym- in the unliganded HMPP kinase structure. Instead, ametrically related counterparts. A notable cluster of aro- different sulfate anion (sulfate 3) is observed along withmatic residues forms a hydrophobic core within the di- sulfate 1 in the unliganded HMPP kinase structure. Sul-mer interface. These include 2-fold-related pairs of fate 3 occupies part of the HMP binding site and formsPhe262, Trp265, and Trp266. The aromatic rings of the hydrogen bonds with Glu44 and with the water moleculeTrp265 pair are approximately parallel to each other that is positioned between Gly11 and Met80.and are separated by about 3.4 A, while the other four The location and approximate mode of ATP and Mg2�

aromatic residues flank the central pair. binding may be deduced by analogy with other riboki-nase family structures determined in the presence of

HMPP Kinase Active Site ATP, ADP, or ATP analogs. The ATP is modeled nearEach HMPP kinase monomer possesses one self-con- the ends of � strands �6, �7, �8, �9, and �10 and helicestained active site that begins near the dimer interface �6 and �8. The modeled ATP is near residues Asp187,and extends outward along the C-terminal edge of the Lys176, Thr211, and Lys237, all of which are highly con-central � sheet. The two active sites in the dimer are served within the family. Based on this model, residuesseparated by approximately 20–25 A. Each active site Lys176 and Thr211 (via water) are expected to interactcontains one HMP binding site and one ATP binding site. with the �-phosphate, and residues Lys176 and Mg2�

The HMP binding site was located using the structure of are expected to interact with the �-phosphate. Otherthe HMPP kinase-HMP complex. In the crystal structure, nearby residues that are highly conserved throughoutthe average B factor for HMP in monomer A is 48.8 A2 the ribokinase family include Asp105 (always Asn orand for HMP in monomer B is 55.1 A2. Repeated attempts Asp), Asn139, Glu142, Thr191 (always Ser or Thr), andto soak or cocrystallize HMPP kinase with ATP or ATP Gly212. The ATP �-phosphate is located near the posi-analogs were unsuccessful. However, the ATP binding tion of sulfate anion 2 of the HMPP kinase-HMP com-site is highly conserved within the ribokinase family, and plex. Loop 199–202 (�10-�8) and loop 178–186 (�8-�9)ATP was easily modeled into the HMPP kinase structure are near the ATP binding site but are disordered in bothusing the ribokinase-ADP, adenosine kinase-AMPPCP, structures of HMPP kinase. The corresponding twoand THZ kinase-ATP complexes. The HMP binding site loops interact with the adenine ring of ATP in otheris near the dimer interface, while the ATP binding site ribokinase family structures [4–7, 9].stretches outwards, with the �-phosphate near the HMPsubstrate and the adenine base farthest from the dimerinterface. Discussion

The HMP binding site involves five � strands (�1, �2,�3, �4, and �5) and three loops (loop 11–19, connecting Comparison with Other Ribokinase

Family Structures�1 and �1, loop 78–83, connecting �5 and �3, and loop45–51, connecting �3 and �4). The first three strands in HMPP kinase is the sixth reported structure that belongs

to the ribokinase family. The monomers of HMPP kinasethe central � sheet (�2, �1, and �5) provide the frame-work for the HMP binding site. Loop 45–51 and an addi- (Protein Data Bank code 1JXH), ribokinase (Protein Data

Bank code 1RK2) [5, 9], human adenosine kinase (Pro-tional loop (109–116) form a flap that acts as a lid forthe active site. In the unliganded structure, loop 109–116 tein Data Bank code 1BX4) [6], T. gondii adenosine ki-

nase (Protein Data Bank code 1DGY) [7], THZ kinaseis disordered and not visible in the electron density

Structure228

Figure 4. The Active Site of HMPP Kinase

(A) Stereoview of the HMP and sulfate ionswith key residues. Atoms are colored codedby atom type (carbon, black; nitrogen, blue;oxygen, red; sulfur, orange). Key hydrogenbonds are indicated by dashed lines.(B) A schematic drawing of the active site.Key hydrogen bonds are indicated by dashedlines.

(Protein Data Bank code 1ESQ) [4], and ADP-dependent sheet domains resulting in an interlocking � clasp, suchthat each active site utilizes residues from both mono-glucokinase (Protein Data Bank code 1GC5) [8] all have

similar folds. Each contains a central � sheet flanked by mers [5]. The four-stranded � sheet forms a lid over theribokinase active site. Such a dimer is not possible inseveral � helices on each side. Comparing the structures

shows that eight � strands and eight � helices are pres- the case of HMPP kinase, because the four-stranded �sheet does not exist. Instead, HMPP kinase has twoent in each fold. Of the 266 amino acids in HMPP kinase,

107 were manually selected for superposition with struc- short � strands (�3 and �4) and a loop (109-116) thatform an active site flap, which closes when the substrateturally equivalent residues in the other five structures

using the computer program ProFit [10] (Figure 5). The is bound to the protein. The active site lid of ribokinasealso opens and closes upon substrate binding but iscomparison showed that human adenosine kinase and

T. gondii adenosine kinase are the most similar pair, well ordered in both open and closed forms [9].Adenosine kinase is monomeric; nevertheless, its ac-with an rmsd of 0.86 A, while the most dissimilar pairs

are THZ kinase and either human adenosine kinase or tive site is also shielded by a lid. In this case, the lidconsists of a five-stranded � sheet flanked by two �glucokinase, both with an rmsd of 2.1 A (Table 1). The

rmsd for HMPP kinase compared with any of the other helices. Like ribokinase, the active site opens and closesupon substrate binding. The active site lid of ADP-depen-five enzymes is 1.6–1.8 A.

Although the monomers of the ribokinase family have dent glucokinase is made from five � strands and four� helices. THZ kinase is trimeric but shows no evidencesimilar folds, the quaternary structures for the active

forms of the enzyme are distinctly different [4–8]. Human of an active site flap. Instead of a flap, its active site isshielded by an adjacent subunit. The subunit contactsand T. gondii Adenosine kinases and ADP-dependent

glucokinase are monomeric, HMPP kinase and riboki- in THZ kinase are provided primarily by several � helicesthat are inserted at the C-terminal end of the polypeptidenase are dimeric, and THZ kinase is trimeric. The dimers

formed by ribokinase and HMPP kinase are also dis- chain.tinctly different. HMPP kinase forms a dimer with a largecontact surface and extensive hydrogen bonding and ATP Binding Site

The nucleotide binding site of HMPP kinase has highvan der Waals interactions. Each monomer in HMPPkinase has a separate active site. The ribokinase dimer structural similarity to the other four structures of ATP-

dependent members in the ribokinase family. Therefore,interface forms between two small, four-stranded �

Structure of HMPP Kinase229

Mg2�/ATP. These include Lys176, Asp187, Arg202, andLys237. Two loops in the nucleotide binding site (resi-dues 178–186 and residues 199–201) are disordered inboth the unliganded and complexed HMPP kinase. Inthe other structures, the corresponding loops are or-dered and participate in binding the adenine moiety ofATP. Therefore, it is likely that these loops would beordered in HMPP kinase after ATP binds.

The recent structure of ADP-dependent glucokinase[8] provides an interesting comparison (Figure 6A) ofnucleotide binding sites. Compared to the ATP-depen-dent ribokinase family members, the terminal �-phos-phate of ADP overlaps with the terminal �-phosphate ofATP for the other family members, and it is the adenosinemoiety that is shuffled along the active site cleft. Thus,the main catalytic apparatus is preserved, while provid-ing alternate adenine and ribose binding sites.

Residues 210–213 (GTGC) in HMPP kinase form thepredicted kinase anion hole [7]. This region is highlyconserved in HMPP kinase from different species. Fur-thermore, it is also the most highly conserved regionamong the entire ribokinase family of proteins. In theHMPP kinase-HMP complex, a sulfate ion was observedFigure 5. Superposition of Ribokinase Family Members Based onnear this site in a position corresponding to the �-phos-107 Structurally Conserved C� Positionsphate of the modeled ATP. In addition to being nearConserved elements from all family members are shown using thick

lines. Nonconserved secondary structure elements for HMPP kinase the anion hole, the sulfate ion also forms a favorableare shown by a thin red line, with main chain breaks connected by hydrogen bond with the HMP hydroxyl group, which isdashed lines. Conserved secondary structural elements are labeled the acceptor of the �-phosphate during the reaction. Inusing HMPP kinase notation; nonconserved elements are not la- the unliganded structure, only a water molecule is pres-beled. Colors: HMPP kinase, red; T. gondii AK, green; glucokinase,

ent at this position, suggesting that the hydrogen bondblue; human AK, aqua; ribokinase, purple; thiazole kinase, yellow.with HMP may play an important role in ATP bindingand alcohol activation.

the location and approximate mode of nucleotide andMg2� binding may be deduced by analogy to structures Substrate Binding Site

The hydroxymethyl group of a small molecule is theof other ribokinase family members that were deter-mined in the presence of adenosine [6], ADP [5, 9], ATP substrate for each of the members of the ribokinase

family. Superposition of the five structures shows that,[4], or ATP analogs [7]. When these structures are super-imposed, a common nucleotide binding site is apparent although there are dramatic differences in the specific

amino acid residues in this site, the overall geometry of(Figure 6A). The ATP binding site is located in a shallowgroove that stretches along the C-terminal edge of the the binding site is similar and the hydroxymethyl groups

are positioned in nearly identical locations (Figure 6B).central � sheet. The positions of the adenine rings andthe �- and �-phosphates are very similar between the This is consistent with the similarity between the ATP

binding sites and the requirement that the ATP �-phos-four examples. The predicted position of the �-phos-phate is less certain, because only the T. gondii adeno- phate be near the hydroxymethyl acceptor group. Al-

though the substrate binding sites are similar, each en-sine kinase complex contains both its substrate (adeno-sine) and an ATP analog. In the crystal structures of zyme utilizes a different mechanism for shielding the

catalytic site from the solvent, as described above. TheHMPP kinase, two well-ordered sulfate ions indicatepossible positions for the �-phosphate group. One of substrate HMP is buried in the active site, with �3, �4,

and loop 109–116 forming the flap that covers it. Analysisthese corresponds to the �-phosphate of the T. gondiiadenosine kinase complex. The nucleotide binding site of the protein surface showed that bound HMP has an

accessible area of about 1 A2 (0.96 A2 in monomer Aof HMPP kinase is lined with several conserved residuesthat may form favorable electrostatic interactions with and 1.32 A2 in monomer B) [11]. The lack of solvent

Table 1. The RMSD in A for the Superposition among Members of the Ribokinase Family Based on 107 Amino Acids

RK AK (Human) AK (T. gondii) THZ Kinase HMP Kinase Glucokinase

RK — 1.0 1.1 2.0 1.6 1.5AK (human) — 0.8 2.1 1.7 1.6AK (T. gondii) — 2.0 1.7 1.4THZ kinase — 1.6 2.1HMPP kinase — 1.8Glucokinase —

Structure230

Figure 6. Comparison of Active Sites of theVarious Ribokinase Family Members

HMPP kinase backbone, grayscale; ligandsfrom HMPP kinase, red; T. gondii AK, green;glucokinase, blue; human AK, aqua; riboki-nase, purple; thiazole kinase, yellow.(A) Superposition of ligands in the ATP or ADPbinding sites. Ligands are ATP from thiazolekinase, ADP from ribokinase and glucoki-nase, AMP-PCP from T. gondii AK, and aden-osine from human AK. For HMPP kinase, themodeled position of ATP is shown. For ATP-dependent enzymes, the adenine base,�-phosphate, and �-phosphate superimposemost closely. Greater variation is seen in theposition of the �-phosphate, especially in thecase of thiazole kinase, for which ATP wasbound in the presence of product monophos-phate (see text for discussion). In glucoki-nase, the ADP shifts so that the �- and�-phosphates overlap with the � and � phos-phate sites of the ATP-dependent enzymes.(B) Superposition of substrates. The hydroxylgroup accepting the phosphate is shown asa large sphere, with the rest of the substraterepresented by bonds only. Substrates areshown for HMPP kinase (HMP), T. gondii AK(adenosine), human AK (adenosine), thiazolekinase (hydroxyethylthiazole), and ribokinase(ribose).

accessibility suggests that water is not needed to facili- dent phosphorylation of HMP-P to form HMP-PP. Usingthe position of HMP observed in the HMPP kinase-HMPtate the deprotonation of HMP.complex and the position of ATP deduced by analogyHMP is bound to the enzyme active site through sev-to nucleotide binding in other members of the ribokinaseeral stabilizing interactions (Figure 4). Glu44 forms afamily, we constructed a model for the enzyme-HMP-hydrogen bond with N4 and a van der Waals interactionATP-Mg2� complex (Figure 7). Based on similarities towith N3. A well-ordered water molecule is bound byadenosine kinase, ribokinase, and THZ kinase, the phos-hydrogen bonds to Gly11 (NH), Met80 (NH), and HMPphorylation of HMP most likely proceeds by an inline(N1). Other residues that form hydrophobic contactsdisplacement mechanism [12]. The model places thewith HMP include Ala18, Val42, Met80, Val107, andsubstrate and ATP molecules in the correct geometryCys213. The distances between the methyl group ofand shields the active site from the solvent.HMP and the protein atoms around it are 3.3–3.9 A.

The reaction would also be facilitated by the presenceThe structure of HMPP kinase complexed to HMPof a base to activate the hydroxymethyl group and analso provides some possible insight into the preferredanion hole to stabilize the transition state. The modelorder of substrate binding. It is unlikely that HMP willindicates that the 7-hydroxyl group of HMP is 3.8 A frombind after ATP, since ATP would block the HMP bindingthe �-phosphate of ATP, and the geometry is consistentsite. Also, it is possible that the presence of HMP facili-with an inline displacement (Figure 7A). The postulatedtates ATP binding by forming hydrogen bonds betweenMg2� ion simultaneously coordinates with both the �-its hydroxyl group and the �-phosphate of ATP. HMPand �-phosphates of the ATP model, such that it shouldis also close to sulfate 2, with distances between thedecrease negative charge, thereby stabilizing ADP as ahydroxyl group and O1 and O3 of 3.0 A and 3.2 A, respec-leaving group and enhancing the reactivity of the �-phos-tively. Since HMP has no strong basic residue aroundphate (Figure 7B). The Mg2� ion also coordinates to thethe oxygen, it is possible that the �-phosphate maycarbonyl oxygen and hydroxyl group of Thr208, whichoccupy this site and function as the alcohol-activatingis highly conserved among HMPP kinase sequences.base in a type of substrate-assisted catalysis that wasThe electrophilicity of the �-phosphate of ATP is alsopreviously proposed for THZ kinase [4].enhanced by hydrogen bonding between a secondphosphate oxygen and the amide proton of Gly210 and

Mechanism of HMPP Kinase by hydrogen bonding between a third phosphate oxygenHMPP kinase catalyzes both the ATP-dependent phos- and the hydroxyl proton of HMP. Gly210 is conserved

throughout the entire ribokinase family and contributesphorylation of HMP to form HMP-P and the ATP-depen-

Structure of HMPP Kinase231

Figure 7. Proposed Mechanism for the TwoActivities of HMPP Kinase

(A) Enzyme-substrate complex for the firstphosphorylation reaction (HMP � ATP ↔HMP-P �ADP).(B) Products for the first phosphorylation re-action.(C) Alternate conformation for the HMP-P inwhich the phosphate group is repositionedfor the second phosphorylation reaction.(D) Enzyme-substrate complex for the sec-ond phosphorylation reaction (HMP-P �

ATP ↔ HMP-PP �ADP).(E) Products for the second phosphorylationreaction.

to the anion hole by donating a hydrogen bond from its kinase because of the absence of an acidic residuehydrogen bonded to N1 of the pyrimidine. One possibil-amide hydrogen atom.

In the enzyme-HMP complex, Asp105 and Cys213 are ity is that the �-phosphate of ATP plays an importantrole in the deprotonation. In the model of the enzyme-closest to the hydroxyl group of HMP. Both residues

are highly conserved among HMPP kinases. However, substrate complex, one oxygen atom of the �-phos-phate is 3.0 A away from the hydroxyl group of HMPCys213 is 4.5 A from the hydroxyl group, and Asp105

is 5.1 A from the hydroxyl group. This suggests that and another oxygen atom is 3.3 A away.The most interesting difference between HMPP ki-neither residue is likely to serve as the general base that

activates the hydroxyl group. In the structure of THZ nase and other members of the ribokinase family is thatHMPP kinase catalyzes two separate phosphorylationkinase [4], the residue corresponding to Cys213 is also

a cysteine. Campobasso et al. proposed that, in THZ reactions. The structure of HMPP kinase suggests thatthere is only one pyrimidine binding site and only onekinase, a water molecule forms a hydrogen bond with

cysteine, while cysteine forms a hydrogen bond with nucleotide binding site. Since there is no evidence tosupport any dramatic conformational change in HMPPthe alcohol [4]. However, mutating the cysteine of THZ

kinase to either serine or alanine resulted in an enzyme kinase itself, it is reasonable to postulate that either the�-phosphate of ATP, the phosphate of HMP-P, or boththat retained significant activity [4]. Interestingly, muta-

tion of the THZ kinase cysteine to aspartate resulted must bind in alternate positions for the second phos-phorylation reaction. In the HMP complex, one of thein a 9-fold increase in activity [4]. In ribokinase and

adenosine kinase, the residue corresponding to Cys213 sulfate anions (sulfate 1) is located about 3.7 A awayfrom the HMP hydroxyl group. When HMP-P is modeledis aspartate, which was postulated to be the residue

used to deprotonate the alcohol [5]. In each case, the into the active site, the phosphate fits easily into this siteafter rotation about the C5-C7 bond while the pyrimidinecysteine or aspartate follows the highly conserved three-

residue sequence that corresponds to the anion hole. ring remains fixed (Figure 7C). It is then possible toarrange the reactants for a second phosphorylation re-Like THZ kinase, a water molecule in HMPP kinase brid-

ges Cys213 and a conserved aspartate (Asp23). How- action by binding a second ATP in the original ATPbinding site (Figure 7D), but with the �-phosphate swungever, because the cysteine mutant in THZ kinase retains

significant activity, it is likely, by analogy, that other toward the HMP-P substrate. The new arrangementplaces the �-phosphate of ATP near the phosphate offactors may contribute to the activation of the HMP

hydroxyl group. The pyrimidine amino group is the base HMP-P, such that the product HMP-PP could be formedthrough an inline displacement reaction. The resultingthat deprotonates the thiazolium moiety of thiamin in all

TPP-dependent reactions. It is unlikely that the pyrimi- geometry for the second transition state is reasonableand has the advantage that both reactions utilize fea-dine amino group is functioning as the base for HMPP

Figure 8. Superposition of the Anion HoleRegions of Ribokinase Family Members

The figure illustrates residues that restrict theconformation of the phosphorylated product.Phe170, Gln38, and adenosine are shown inlight blue for human adenosine kinase. Tyr169and adenosine are shown in green for T. gon-dii adenosine kinase. Lys43 and ribose areshown in purple for ribokinase.

Structure232

Figure 9. Topology Diagrams of RibokinaseFamily

Secondary structure elements that are com-mon to all family members are shown in thesame color (central � sheet, blue; � heliceson each side, red and green). The secondarystructural elements in gray represent an in-sertion relative to THZ kinase. The secondarystructural elements in yellow represent an in-sertion relative to HMPP kinase. The second-ary structural elements in black represent aninsertion relative to ribokinase. The second-ary structural elements in light blue representinsertions relative to adenosine kinase. Thestructural elements chosen for assignment toinsertions minimize the total number of inser-tions and conserve edge strands of sheets.The comparison suggests that the enzymesevolved in the following order: THZ kinaseto HMPP kinase to ribokinase to adenosinekinase to glucokinase. The figure was pre-pared with TOPS [40].

tures of the same highly conserved anion hole (Figure while the �-phosphate of the ATP molecule bends outand away from the anion hole and therefore cannot carry7E). Further transition state stabilization may be pro-

vided by positively charged residues, such as Lys176, out the second phosphorylation. Adenosine is con-verted to ADP by two separate enzymes (adenosinewhich has no apparent role in the first phosphorylation

reaction. Thus, the HMPP kinase active site utilizes simi- kinase and adenylate kinase), while ribose 5-phosphateis not converted to ribose 5-diphosphate by any knownlar catalytic strategies for the two phosphorylation reac-

tions by providing overlapping binding sites for the two enzyme. Attempts to model ribose 5-phosphate plusATP into ribokinase or AMP plus ATP into adenosinerelated substrates, HMP and HMP-P.

Given their structural homology, it is reasonable to kinase showed that, in either case, residues with largeside chains anchor the product phosphate group nearconsider why the other ribokinase family members cata-

lyze only the first type of phosphorylation reaction and the anion hole (Phe170 and Gln38 in human AK [6],Tyr169 in T. gondii AK [7], and Lys43 in ribokinase [5])not the second. We postulate that only HMPP kinase

catalyzes a second phosphorylation reaction because (Figure 8). Thus, the only way to accommodate ATP isto release the phosphorylated product.only its active site has enough space to accommodate

the phosphate of the second substrate as well as ATP.Direct evidence is provided by the structure of the THZ Role of HMPP Kinase in Bacimethrin Resistance

Bacimethrin is a natural product isolated from gram-kinase-Thz-P-ATP complex [4]. In this complex, thephosphate group of the product stays in the anion hole, positive bacteria, including Bacillus megaterium [13]

Table 2. Summary of X-ray Diffraction Data Collection and Processing

Native Complex Edge Peak Remote

Wavelength (A) 0.909 0.909 0.9795 0.9793 0.9537Resolution 2.3 2.6 3.1 3.1 3.1Space group P41212 P41212 P41212 P41212 P41212Cell dimensions

a 77.72 77.75 76.55 76.55 76.55b 183.42 183.49 182.68 182.68 182.68

Number of reflections 177,754 186,929 176,984 176,819 176,951Number of unique reflections 25,741 20,166 10,601 10,616 10,599Redundancy 7.0 (7.3) 4.7 (4.8) 9.4 (7.6) 9.2 (9.1) 9.3 (8.9)Completeness (%) 99.9 (99.9) 99.7 (99.6) 99.3 (94.7) 99.1 (93.5) 99.4 (95.9)

0.061Rsym (%) 0.063 (0.217) 0.062 (0.260) 0.057 (0.115) 0.056 (0.089) (0.114)I/� 8.3 (3.5) 10.4 (2.9) 16.6 (11.6) 16.9 (12.7) 16.1 (10.7)

Structure of HMPP Kinase233

of HMPP kinase and inserting two additional � strandsTable 3. Summary of Refinement Statisticsto form a four-stranded � sheet flap. This flap is stabi-

Native Native/HMPlized by dimer formation in which two � sheets pack

Resolution range (A) 25–2.3 25–2.6 back to back. Furthermore, these two � sheets ex-Reflections used 25,741 20,134 change a strand, such that the ATP binding site residuesR factor (%) 23.5 23.0

are contributed by both � sheets. In adenosine kinase,Rfree (%) 27.5 27.0the lid becomes independent by the insertion of an addi-Number of protein atoms 3764 3804tional � strand and two amphipathic � helices. The newNumber of HMP — 2

Number of SO4�2 4 4 � strand replaces the strand that comes from dimer

Number of water molecules 139 75 formation in ribokinase, and the two � helices stabilizeRmsd the five-stranded sheet. In ADP-dependent glucokinase,

Bonds (A) 0.0059 0.0067the lid is also shielded by amphipathic � helices. In thisAngles () 1.24 1.27case, two additional helices are inserted with respectDihedrals () 22.95 23.20to the adenosine kinase lid, giving a total of five � strandsImproper () 0.80 0.87

B factors and four � helices.Average (A2) 41.5 43.6Minimum (A2) 21.9 17.0

Biological ImplicationsMaximum (A2) 98.5 131.4B rmsd

Bonded main chain atoms (A2) 1.27 1.45 In this paper, we have presented the structure of HMPPBonded side chain atoms (A2) 2.08 1.88 kinase, an essential enzyme in the biosynthesis of thia-Angle main chain atoms (A2) 2.09 2.56 min pyrophosphate. HMPP kinase catalyzes two sepa-Angle side chain atoms (A2) 3.10 2.93

rate ATP-dependent phosphorylation reactions withinNCS rmsd (A) 0.019 0.022the pathway: (1) the conversion of HMP to HMP-P and(2) the conversion of HMP-P to HMP-PP. The structureconfirms that HMPP kinase is a homodimer and a mem-

and Streptomyces albus [14], and is toxic to bacteriaber of the ribokinase family. Although the known struc-

and yeast growing on minimal medium. S. typhimuriumtures (ribokinase, adenosine kinase, THZ kinase, and

is unable to grow on minimal medium in the presenceHMPP kinase) have a common monomer fold, they differ

of 130 nM bacimethrin [15]. Bacimethrin has been shownin substrate specificity and quaternary structure. Com-

to be a thiamin antimetabolite [16] and differs from HMPparison of the four folds suggests that divergence of

only by the replacement of the methyl group at the 2substrate specificity and quaternary structure may be

position by a methoxy substituent (CH3O-HMP). HMPPcorrelated with the evolution of an active site lid that

kinase can accept bacimethrin as a substrate. Resis-shields the substrate from the solvent. If this is true,

tance to bacimethrin in Salmonella typhimurium can bethen evolution progressed from THZ kinase (no lid) to

traced to the P186Q and S15N mutations in the HMPPHMPP kinase (flap formed by two short � strands and

kinase gene [15]. Examination of the HMPP kinase struc-connecting loop) to ribokinase (lid formed by four �

ture from Salmonella typhimurium reveals that the S15Nstrands from one subunit and one � strand from the

mutation is near the 2 position of the pyrimidine sub-2-fold-related subunit) to adenosine kinase (lid formed

strate. Therefore, it is likely that the larger asparagineby five � strands and two � helices) and finally to ADP-

side chain inhibits bacimethrin binding. The P186Q mu-glucokinase (lid formed by five � strands and four �

tation is located near the proposed ribosyl group of thehelices). The structure of HMPP kinase also leads to

ATP, and the basis for its effect is less clear. It is possibleideas about how it catalyzes two different phosphoryla-

that this mutation alters the conformation of ATP andtion reactions. Modeling substrates and products into

interferes with one of the phosphorylation reactions.the active site suggests that HMP-P, the product of thefirst reaction and the substrate for the second reaction,Implications for Evolution of thecan bind in two different modes. The pyrimidine ringRibokinase Familybinds approximately in the same way for both modes,

Comparison of the six available structures, representingbut the phosphate group can swing into alternate posi-

four ATP-dependent enzyme activities and one ADP-tions by rotating about the C5-C7 bond. In one mode,

dependent enzyme activity, suggests a possible evolu-the �-phosphate of ATP can be transferred to the alcohol

tionary pathway (Figure 9). The simplest fold that con-of HMP, while in the second mode, the �-phosphate is

tains all the necessary elements for catalytic activitynear the phosphate group of HMP-P and ready to form

is the THZ kinase monomer. THZ kinase contains thethe product of the second reaction, HMP-PP. This

substrate and ATP binding sites as well as the anionscheme allows HMPP kinase to use similar structural

hole. However, THZ kinase lacks a lid for shielding thefeatures to stabilize the transition states of both reac-

active site from solvent. To compensate, THZ kinasetions. Given the widespread use of pyrophosphates in

forms trimers in which each subunit contains one activebiosynthesis, it is surprising that this dual kinase activity

site and the active site is shielded by contacts with thehas been identified in only one other system [17].

adjacent subunit. HMPP kinase lacks a well-defined lidbut instead utilizes a flap formed by a flexible loop and

Experimental Procedurestwo short � strands inserted between �2 and �5. Thesestrands together with the loop form a nascent lid that Protein Productionis stabilized by the dimer interface. In ribokinase, the The thiD gene was excised from the previously described PET16b-

based overexpression plasmid [3] using NdeI and BamHI andlid is fully formed by extending the two short � strands

Structure234

spliced into the T7-based expression vector PET28a, which contains The ten Se atom sites were used for parameter refinement andphase calculation with the MLPHARE program [28]. The figure ofa 5 sequence encoding a shorter polyhistidine tag. The plasmid

was transformed into E. coli strain BL21(DE3). The transformed cells merit (FOM) was 0.568 for 10,464 reflections in the resolution rangebetween 25 and 3.1 A. Calculation of electron density maps con-were grown in LB medium containing 40 mg/l of kanamycin at 37C

until the OD600 reached 0.6. Then, the culture was induced by the firmed P41212, rather than P43212, to be the correct space group.Electron density modification and noncrystallographic symmetryaddition of IPTG to a final concentration of 1 mM, and the tempera-

ture was reduced to 25C. The cells were harvested after overnight (NCS) averaging were performed with the DM program [29]. TheNCS matrix was calculated in O [30] using the coordinates of thegrowth and stored at �80C.

The cell pellet was suspended in 10 times w/v of a buffer con- ten Se atoms. The FOM increased to 0.846 with a free R factor of29.6%.taining 50 mM sodium phosphate (pH 7.8) and 300 mM NaCl (buffer

A), lysed using a French press and equilibrated with Ni-NTA agaroseresin (Qiagen) at 4C for 1 hr. The resin was eluted with buffer A, Model Building and Refinementbuffer A was eluted with 10% glycerol (buffer B), and buffer B was The initial model was built with a 3.1 A resolution electron densityeluted with 10, 20, 40, 60, and 100 mM imidazole. The pure protein map using the computer graphics program O [30]. After initial modelwas in the 60 and 100 mM imidazole fractions. The protein solution building, residues 110–115, 177–186, and 199–204 were omitted duewas buffer exchanged to 50 mM Tris-HCl containing 1mM dithi- to the poor electron density. The N-terminal histidine tag was alsoothreitol and concentrated. Selenomethionyl HMPP kinase for MAD invisible in the density. All the model refinements were carried outphasing was expressed using E. coli BL834 (DE3) [18]. The purifica- using the program CNS [31]. An NCS restraint was generated basedtion procedure for the selenomethionyl HMPP kinase was essentially on the C� coordinates and applied in the refinement. After one roundthe same as that for the native enzyme. of refinement, the R factor and the Rfree converged to 24.9% and

32.0%, respectively.The 2.3 A resolution unliganded HMPP kinase data were used forCrystallization

further refinement. The high-resolution data were gradually includedCrystallization conditions were screened using Crystal Screen Kitsat the rate of 0.2 A per round. As the refinement proceeded, newI and II (Hampton Research). Further optimization resulted in crystal-electron density corresponding to residues 202–204 and two sulfatelization conditions based on a hanging drop vapor diffusion setupions in each monomer were clearly revealed in Fo � Fc electronat 18C. Crystals were obtained with and without HMP under similardensity maps. In addition, 139 water molecules were introduced.conditions using 6 mg/ml HMPP kinase. The hanging drops con-The R factor and Rfree for the final model are 23.5% and 27.5%,tained 2 �l protein solution and 2 �l reservoir solution. Crystalsrespectively. This model has 496 residues, 4 sulfate ions, and 139without HMP were obtained using a reservoir solution containingwater molecules. The Ramachandran plot [32] showed 90.5% of the1.35 M MgSO4 and 0.15 M MES (pH 7.0). Crystals with HMP wereresidues in the most favored region and 9.5% in additional allowedobtained using a reservoir solution containing 1.4 M MgSO4 andregions. The Luzzati plot [33] estimated the coordinate error to be0.15 M MES (pH 7.2). Crystals of selenomethionyl HMPP kinase0.31 A from the working set and 0.36 A from the test set.were obtained using 8 mg/ml SeMet HMPP kinase and a reservoir

The structure of the HMPP kinase-HMP complex was determinedsolution containing 1.6 M MgSO4, 0.15 M MES (pH 7.0), and 2.2%by refining the unliganded HMPP kinase model against the data for1,6-hexanediol. All crystals belong to space group P41212 with ap-the complex. HMP was clearly defined in a difference electron den-proximate unit cell dimensions of a � 77 A and c � 183 A. Attemptssity map and manually fitted. Bond distance and angle restraintsto crystallize HMPP kinase with various analogs of ATP, with andfor HMP were adapted from a thiamin phosphate structure foundwithout substrate, were unsuccessful.at the Hetero-Compound Information Centre-Uppsala [34]. In thedifference map based on the HMP complex data set and the unli-X-Ray Data Collection and Processingganded HMPP kinase data set, the density for the backbone fromMonochromatic X-ray intensity data were measured for unliganded110 to 115 was clearly revealed. The B factors for these residuesHMPP kinase and the HMPP kinase-HMP complex at cryogenicwere between 74 and 129 A2 after introducing the correspondingtemperatures on station A1 at the Cornell High Energy Synchrotronresidues in the structure. Multiple rounds of simulated annealingSource (CHESS). All data were measured with an Area Detectorand individual temperature factor refinement were carried out toSystems Corporation Quantum 4 CCD detector, placed 200 mmimprove the model. Final refinement statistics are given in Table 3.from the sample. To freeze the crystals, three reservoirs containing

well solution plus 10%, 16%, or 24% glycerol, respectively, wereAcknowledgmentsused. Crystals were equilibrated with each freezing solution for 1

min, moving from lowest to highest glycerol concentration, thenWe thank the Structural Biology Center (beamline ID-19) of the Ad-flash frozen in liquid nitrogen. A total of 93 and 60 of data werevanced Photon Source and the Cornell High Energy Synchrotronmeasured with the oscillation method for native HMPP kinase andSource for providing beam time for the studies. We thank Drs. QuanHMPP kinase-HMP complex, respectively. The intensity data wereHao, Irimpan Mathews, and Todd Appleby for assistance with dataintegrated with MOSFLM [19] and scaled and merged with SCALAcollection and analysis and Ms. Leslie Kinsland for assistance inin the CCP4 package [20, 21]. The output file was edited using thethe preparation of this manuscript. This work was supported by NIHTRUNCATE program in the CCP4 package [22]. Details of the datagrants RR-01646 and DK44083 (to T.P.B.), the W.M. Keck Founda-collection and processing are given in Table 2.tion, and the Lucille P. Markey Charitable Trust.The multiple wavelength anomalous diffraction (MAD) data were

measured at cryogenic temperature on beam line 19ID at the Ad-vanced Photon Source (APS). Three wavelengths were selected Received: September 17, 2001for data collection, corresponding to the maximum f″ (peak), the Revised: December 11, 2001minimum f (edge), and a reference wavelength (remote). A total of Accepted: December 13, 2001three times, 73 of data were measured using a 1 oscillation anglefor 60 s. The data were processed and scaled using DENZO and ReferencesSCALEPACK [23]. Details of the data collection and processing aregiven in Table 2. 1. Begley, T.P., Downs, D.M., Ealick, S.E., McLafferty, F.W., Van

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