doi:10.1016/j.jmb.2004.01.043 J. Mol. Biol. (2004) 337, 387–398 Substrate Specificity and Mechanism from the Structure of Pyrococcus furiosus Galactokinase Andrew Hartley1, Steven E. Glynn1, Vladimir Barynin1 Patrick J. Baker1, Svetlana E. Sedelnikova1, Corne´ Verhees2 Daniel de Geus2, John van der Oost2, David J. Timson3 Richard J. Reece4 and David W. Rice1* 1Krebs Institute, Department Galactokinase (GalK) catalyses the first step of the Leloir pathway of of Molecular Biology and galactose metabolism, the ATP-dependent phosphorylation of galactose Biotechnology, University to galactose-1-phosphate. In man, defects in galactose metabolism can of Sheffield, Sheffield S10 2TN result in disorders with severe clinical consequences, and deficiencies in UK galactokinase have been linked with the development of cataracts within the first few months of life. The crystal structure of GalK from Pyrococcus 2Laboratory of Microbiology furiosus in complex with MgADP and galactose has been determined to Wageningen University 2.9 A˚ resolution to provide insights into the substrate specificity and cata- Hesselink van Suchtelenweg 4 lytic mechanism of the enzyme. The structure consists of two domains NL-6703 CT Wageningen, The with the active site in a cleft at the domain interface. Inspection of the sub- Netherlands strate binding pocket identifies the amino acid residues involved in galac- 3Medical Biology Centre tose and nucleotide binding and points to both structural and mechanistic School of Biology and similarities with other enzymes of the GHMP kinase superfamily to which Biotechnology, Queen’s GalK belongs. Comparison of the sequence of the Gal3p inducer protein, University Belfast, 97 Lisburn which is related to GalK and which forms part of the transcriptional Road, Belfast BT9 7BL, UK activation of the GAL gene cluster in the yeast Saccharomyces cerevisiae, 4 has led to an understanding of the molecular basis of galactose and School of Biological Sciences nucleotide recognition. Finally, the structure has enabled us to further University of Manchester our understanding on the functional consequences of mutations in 2.205 Stopford Building human GalK which cause galactosemia. Oxford Road, Manchester M13 q 2004 Elsevier Ltd. All rights reserved. 9PT, UK Keywords: galactokinase deficiency; GHMP kinase superfamily; Pyrococcus *Corresponding author furiosus; X-ray crystallography Introduction utilises the glycolytic pathway as a main catabolic route. Conversion of galactose via glycolysis Pyrococcus furiosus is a hyperthermophilic requires an additional metabolic branch, the Leloir archaeon, isolated from thermally active regions. pathway,1 and the first step of this pathway Like other saccharolytic organisms, P. furiosus involves the conversion of galactose to galactose- 1-phosphate, catalysed by galactokinase (GalK; EC g Abbreviations used: FA, structure factor amplitudes for 2.7.1.6), which drives the transfer of the -phos- the anomalous scatterers; GalK, galactokinase; GHMP, phate group from ATP to the O1 position of galactokinase homoserine kinase, mevalonate kinase galactose. phosphomevalonate kinase; GTP, guanosine-50- In man, galactosemia is an autosomal recessive triphosphate; HSK, homoserine kinase; MAD, multi- disorder caused by a defect in one of the three wavelength anomalous dispersion; MMK, Methanococcus Leloir pathway enzymes involved in galactose janaschii mevalonate kinase; NCS, non-crystallographic metabolism, galactokinase, galactose-1-phosphate symmetry; Pf, Pyrococcus furiosus; RMK, Rattus norvegicus uridyl transferase (GalT) and UDP-galactose 40 mevalonate kinase; rmsd, root-mean-squared deviation; 2 SeMet, selenomethionine; s, standard deviation. epimerase (GalE). Deficiency in the transferase, E-mail address of the corresponding author: GalT, is responsible for classic galactosemia (MIM d.rice@sheffield.ac.uk 230400) and can lead to severe neonatal symptoms 0022-2836/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. 388 Structure of P. furiosus Galactokinase Figure 1. Structure-based sequence alignment of representative members of the GHMP family, galactokinase family and yeast Gal3p. GalK, galactokinase; HSK, Methanococcus janaschii homoserine kinase; RMK, Rattus norvegicus mevalo- nate kinase; MMK, M. janaschii mevalonate kinase; Pf, Pyrococcus furiosus; Hs, Homo sapiens; Ec, Escherichia coli; Sp, Structure of P. furiosus Galactokinase 389 such as failure to thrive, hepatomegaly and the first to be solved for any member of this family bacterial sepsis. Galactokinase deficiency (galacto- and reveals a novel nucleotide binding fold and a semia II; MIM 230200) in man is an inborn error of less common, syn conformation of the glycosidic galactose metabolism and is linked to development bond of the ADP in the active site.20 However, of cataracts during the first months of life and also determination of the structure of another GHMP pre-senile cataracts, the onset of which is between kinase family member, mevalonate kinase,16 in 20 and 50 years of age.3 Over 20 mutations have complex with ATP, revealed that in some enzymes been identified in human galactokinase that are of the family, the glycosidic bond can adopt an associated with reduced galactokinase activity in anti conformation. Analysis of the structures of the the blood,4–8 11 of these mutations are single GHMP kinase superfamily has shown that the amino acid substitutions and the biochemical most conserved motif across the GHMP kinase characteristics of some of these have recently been family, with a consensus of PX3GL(G/S)SSA characterised.9 (motif 2; Figure 1), forms an atypical phosphate Studies on the yeast Saccharomyces cerevisiae have binding P-loop which interacts through hydrogen led to identification of a galactokinase (Gal1p) in bonds to the a and b-phosphate groups of ADP. this organism as part of a gene cluster which More recently, the three-dimensional structure of encodes the enzymes of the Leloir pathway.10,11 the GalK from Lactobacillus lactis in complex with The transcriptional activation of this cluster, which galactose and inorganic phosphate has been results in Gal1p expression involves three proteins: reported. This structure confirms the general simi- a transcriptional activator and DNA-binding larity of the GalK fold to the other members of the protein, Gal4p; a repressor, Gal80p; and a ligand GHMP kinase superfamily.21 sensor and inducer, Gal3p. Induction occurs as a Here, we report the three-dimensional structure result of an ATP and galactose-dependent inter- of Pf GalK at 2.9 A˚ resolution in a complex with action between Gal3p and Gal80p with Gal3p ADP and galactose. The structure reveals the acting as the ligand sensor and transducer of galac- mode of nucleotide recognition and its relationship tose signal.12 Sequence analysis has shown that to the galactose-binding site. Comparison of the there is a very high similarity (73% identity) structure with other members of the GHMP kinase between the yeast proteins Gal1p and Gal3p.13 superfamily has permitted the identification of the However, despite this similarity, Gal3p does not residues involved in substrate binding and show galactokinase activity and the transcriptional catalysis and led to a better understanding of the activation of the gene cluster by Gal1p is also lack of galactokinase activity in Gal3p. The observed, although at a much reduced level com- sequence similarity between Pf GalK and human pared to Gal3p.13 Determination of the structure of GalK has facilitated a homology-based modelling Pyrococcus furiosus (Pf) GalK may offer insights study, which has shed light on the structural basis into the mechanism of galactose and ATP binding of the galactosemia causing amino acid substi- in the yeast Gal3p protein and also shed light on tutions in the human GalK protein. the interactions involved in the Gal3p–Gal80p complex. Sequence similarities have suggested that GalK Results and Discussion is a member of the GHMP kinase superfamily14 for which the structures of several family members Quality of the structure have now been reported, including homoserine kinase from Methanococcus janaschii15 (HSK; EC The GalK structure was solved using multi- 2.7.1.39), mevalonate kinase from Rattus wavelength anomalous dispersion (MAD) data norvegicus16 (RMK; EC 2.7.1.36), and from collected on a single selenomethionine labelled M. janaschii17 (MMK), mevalonate diphosphate (SeMet) crystal to 2.9 A˚ resolution. The final decarboxylase from M. janaschii18(MDD; EC refined model of GalK contains nine independent 2.7.4.2) and phosphomevalonate kinase from subunits in the asymmetric unit (labelled A Streptococcus pneumoniae19 (PMK; EC 2.7.4.2). through I in Figure 2(a)), a total of 2999 amino Although the overall sequence identity in the acid residues. Analysis of the packing in the cell family is low20 (with 16%, 15% and 20% identities reveals subunit I is related by a crystallographic between Pf GalK and HSK, RMK and MMK, 2-fold axis to its symmetry related partner, I0 and respectively), three conserved glycine-rich motifs subunits A and D appear to be related by an can be identified (motifs 1–3; Figure 1). The three- approximate non-crystallographic 2-fold axis of dimensional structure of homoserine kinase was symmetry within the crystal. However, the Streptococcus pneumoniae; Sc, Saccharomyces cerevisiae. The invariant residues are highlighted in black with identities in 7/9 sequences highlighted in dark grey. Identities in 3/4 of the non-yeast galactokinases are highlighted in light grey.