中国科技论文在线 http://www.paper.edu.cn

Comparative Biochemistry and Physiology, Part D 3 (2008) 96–107

Molecular evolution of the vertebrate family: Identification of a conserved fifth vertebrate hexokinase gene ⁎ David M. Irwin a, , Huanran Tan b

a Department of Laboratory Medicine and Pathobiology, Banting and Best Diabetes Centre, University of Toronto, Toronto, Canada b Department of Pharmacology, Peking University Health Sciences Centre, Beijing, China Received 6 October 2007; received in revised form 12 November 2007; accepted 13 November 2007 Available online 3 December 2007

Abstract

Hexokinases (HK) phosphorylate sugar immediately upon its entry into cells allowing these sugars to be metabolized. A total of four have been characterized in a diversity of vertebrates—HKI, HKII, HKIII, and HKIV. HKIV is often called (GCK) and has half the molecular weight of the other hexokinases, as it only has one hexokinase domain, while other vertebrate HKs have two. Differing hypothesis has been proposed to explain the diversification of the hexokinase gene family. We used a genomic approach to characterize hexokinase in a diverse array of vertebrate species and close relatives. Surprisingly we identified a fifth hexokinase-like gene, HKDC1 that exists and is expressed in diverse vertebrates. Analysis of the sequence of HKDC1 suggests that it may function as a hexokinase. To understand the evolution of the vertebrate hexokinase gene family we established a phylogeny of the hexokinase domain in all of the vertebrate hexokinase genes, as well as hexokinase genes from close relatives of the vertebrates. Our phylogeny demonstrates that duplication of the hexokinase domain, yielding a HK with two hexokinase domains, occurred prior to the diversification of the hexokinase gene family. We also establish that GCK evolved from a two hexokinase domain-containing gene, but has lost its N-terminal hexokinase domain. We also show that parallel changes in enzymatic function of HKI and HKIII have occurred. © 2007 Elsevier Inc. All rights reserved.

Keywords: Glucokinase; ; Hexokinase; Gene evolution

1. Introduction 1995, 2003; Cárdenas et al., 1998). Hexokinase IV is more often called glucokinase (GCK) although it does not differ in its of by ATP occurs immediately specificity to glucose compared to the other hexokinases upon its import into eukaryotic cells, with the phosphorylation (Wilson, 1995, 2003; Cárdenas et al., 1998). Most hexokinases being mediated by hexokinases (HK) (Wilson, 1995, 2003; are widely expressed but distinct hexokinases dominate specific Cárdenas et al., 1998). Hexokinases phosphorylate a variety of tissues (Katzen and Schimke, 1965, Rogers et al., 1975). HKI is hexose sugars, although their preferred substrate is glucose the most abundant hexokinase in brain and kidney (Griffin (Wilson, 1995). Phosphorylated sugars become substrates for et al., 1992). HKII is the primary hexokinase in muscle and differing metabolic fates, dependent upon the cell type and its adipose tissues (Postic et al., 1994). HKIII predominates in metabolic status (Wilson, 1995, 2003; Cárdenas et al., 1998). A spleen and lymphocytes (Coerver et al., 1998). Glucokinase total of four hexokinases have been identified and extensively (HKIV) is the major hexokinase in glucose sensitive tissues studied in vertebrates, hexokinase I, II, III, and IV (Wilson, such as liver and pancreatic tissues (Printz et al., 1993). In addition to the well-characterized hexokinases, an expressed gene has been identified in mammalian genomes that encode a ⁎ Corresponding author. Department of Laboratory Medicine and Pathobiol- ogy, University of Toronto, 100 College St., Toronto, Ont., Canada M5G 1L5. product that has ADP-dependent glucokinase activity, although Tel.: +1 416 978 0519. this activity has not been demonstrated in vivo (Ronimus and E-mail address: [email protected] (D.M. Irwin). Morgan, 2004), thus its role in glucose is unclear.

1744-117X/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.cbd.2007.11.002 转载 中国科技论文在线 http://www.paper.edu.cn D.M. Irwin, H. Tan / Comparative Biochemistry and Physiology, Part D 3 (2008) 96–107 97

Mammalian hexokinases I, II, and III are approximately explain the evolution of glucokinase, but also help explain the 100 kDa in size, while glucokinase (HKIV) is only half of this diversification of this gene family and provide insights into how size at only 50 kDa (Wilson, 1995, 2003; Cárdenas et al., 1998). the changes in enzymatic function of these have Hexokinases from non-vertebrate animals, as well as plants and occurred. fungi are similar in size to glucokinase (Wilson, 1995, 2003; Here we report a characterization of the hexokinase gene Cárdenas et al., 1998). These observations had suggested that family in vertebrates with an analysis of their evolutionary the 100 kDa mammalian hexokinases were the product of a history. Surprisingly we found five, not four, types of duplication of an ancestral 50 kDa hexokinase (Colowick, 1973; hexokinase-like genes. All five of these hexokinase genes Easterby and O'Brien, 1973). Subsequent analysis of the amino were found throughout vertebrates, and in similar genomic acid sequences of hexokinases revealed extensive sequence locations, suggesting that they were products of gene duplica- similarity between the proteins, and clearly demonstrated that tion events that occurred very early in vertebrate evolution. the hexokinase I, II, and III sequences possess duplicated Multiple hexokinase sequences were also found in non- hexokinase domains, and likely were descendent from a gene vertebrate species, including sea urchin and two species of generated by the duplication and fusion of a single hexokinase Ciona, species that are closely related to vertebrates. None of domain-containing ancestor (Griffin et al., 1991; Kogure et al., the non-vertebrate species, though, encoded a hexokinase 1993; Cárdenas et al., 1998). The existence of two hexokinase encoding more than one hexokinase domain. Phylogenetic domains in the HK1, HKII, and HKIII enzymes suggested the analysis of vertebrate and non-vertebrate hexokinases showed possibility that each of these hexokinases may have two active that all the vertebrate sequences were more closely related to sites. Only HKII has been shown to have two active sites (Tsai each other than to any other species, suggesting independent and Wilson, 1996), as the N-terminal hexokinase domains of duplications of the hexokinase genes on multiple lineages, and both HKI and HKIII do not have catalytic activity (White and that the 100 kDa hexokinase originated on the vertebrate lineage Wilson, 1989; Tsai and Wilson, 1997; Tsai, 1999). The after divergence from non-vertebrate species. Our phylogenetic determination of the crystal structure of hexokinase I (Aleshin analysis also showed that the ancestor of all vertebrate et al., 1998a,b) and glucokinase (Kamata et al., 2004) together hexokinases contained two hexokinase domains, thus glucoki- with molecular modeling and site-directed mutagenesis studies nase did lose its N-terminal hexokinase domain. Our phylogeny of glucokinase (Xu et al., 1994, 1995; Mahalingam et al., 1999; also shows that the N-terminal hexokinase domains of HKI and Marotta et al., 2005) have led to a better understanding of the HKIII independently lost hexokinase activity. amino acid residues essential for catalytic activity. While it is clear that HKI, HKII, and HKIII are descendent 2. Materials and methods from a common ancestor that was generated by the duplication of a single hexokinase domain-containing gene, the relationship 2.1. Molecular databases and sequence analysis of glucokinase to these two hexokinase domain-containing hexokinases, as well as the relationships among these Protein databases maintained by the National Center for hexokinases are unclear. Previous sequence analyses have Biotechnology Information (NCBI GenBank, www.ncbi.nlm. yielded support for two different evolutionary histories. A nih.gov) were searched using the BLASTP algorithm (Altschul phylogenetic analysis by Griffin et al. (1991) suggested that the et al., 1997) for sequences similar to human hexokinases I, II, C-terminal hexokinase domains of hexokinases I, II, and III are III, and IV (GCK). Genomic databases of diverse vertebrate more closely related to glucokinase than they were to the N- species (see Table 2) maintained by Ensembl (version 45, www. terminal hexokinase domains of these hexokinases. This ensembl.org) and NCBI were then searched for gene sequences relationship would suggest that glucokinase is descendent similar to human hexokinase genes using the tBLASTN from a hexokinase that contained two hexokinase domains, but algorithm (Altschul et al., 1997). Gene sequences were that it lost its N-terminal hexokinase domain after it diverged downloaded and used for reciprocal BLAST searches to confirm from the other hexokinases (Griffin et al., 1991; Tsai and that they were more similar to human hexokinase genes than to Wilson, 1996). In contrast, Cárdenas et al. (1998) concluded any other gene. Hexokinase sequences from non-vertebrate from their phylogenetic analysis that glucokinase diverged from hexokinase species (i.e., Ciona intestinalis, Ciona savignyi, and the ancestor of the other hexokinase genes prior to the Strongylocentrotus purpuratus) were used to search the duplication of the hexokinase domain (Cárdenas et al., 1998). vertebrate genomes to exclude the possibility that hexokinase This evolutionary history suggests that the glucokinase gene, genes exist in some vertebrates and that orthologs of those and its ancestors, never possessed two hexokinase domains, and genes do not exist in humans. No such gene was identified. The its structure has not changed since its divergence from other syntenic arrangement of genes adjacent to hexokinase genes hexokinases (Cárdenas et al., 1998). The two evolutionary was determined using gene maps from Ensembl or NCBI. hypotheses differ considerably for the evolutionary history of glucokinase, one suggests that little has change to structure has 2.2. Sequence alignment and phylogenetic analysis occurred, while the other suggests that glucokinase had an ancestor that had two hexokinase domains, but has lost its N- Protein sequences for hexokinase and hexokinase-like genes terminal hexokinase domain. A better understanding of the from diverse vertebrate species and close relatives (see Tables 2 evolutionary relationships of the hexokinases should not only and S1) were downloaded from GenBank and Ensembl. Protein 中国科技论文在线 http://www.paper.edu.cn 98 D.M. Irwin, H. Tan / Comparative Biochemistry and Physiology, Part D 3 (2008) 96–107 sequences were aligned using Clustal W (Thompson et al., macaque genome, nor in any other mammal examined, 1994) either as implemented at the European Bioinformatics indicating that the pseudogene was inserted into the genome Institute (EBI) web server (http://www.ebi.ac.uk/Tools/ on the human lineage between 5 and 25 million years ago, clustalw/) or within MEGA 4 (Tamura et al., 2007). Initial before the human–chimpanzee divergence but after the Old- alignments were conducted using the EBI site, while MEGA World monkey–human divergence. was used to generate alignments used for phylogenetic In addition to the five previously identified hexokinase-like analyses. Both Clustal W at EBI and within MEGA generated sequences characterized in the , we found a sixth similar protein alignments. Phylogenetic relationships of the human hexokinase-like gene adjacent to the HKI gene on hexokinases or the hexokinase domains were estimated using human 10 (Table 1, Fig. 1). This gene has been the neighbor-joining distance method, using either PAM (point named hexokinase domain-containing 1 (HKDC1) within the accepted mutation) or JTT (Jones, Taylor, Thornton) distances human genome databases. The HKI and HKDC1 genes are and by parsimony as implemented in MEGA 4 (Tamura et al., arranged head-to-tail suggesting that they are products of a 2007). tandem gene duplication event. Searches of the NCBI EST databases indicate that HKDC1 is expressed, and since its 3. Results and discussion sequence predicts an intact open reading frame of 917 amino acids it may be functional (see Section 3.4 below). Thus there 3.1. Human genome contains six hexokinase-like genes appears to be five intact, and potentially functional, hexokinase- like genes in the human genome. This observation appears to be To begin to understand the relationships between, and the in conflict with previous survey of hexokinase activity in origin of, the vertebrate hexokinase genes we searched the diverse vertebrates, where activity of several genes could not be human genome for hexokinase-like gene sequences. As found (e.g., see Ureta et al., 1973, 1981). While we have found expected the previously characterized human HKI (Ruzzo the genes for these enzymes, we have not examines the sites or et al., 1998), HKII (Malkki et al., 1994), HKIII (Sebastian et al., levels of expression of these genes (except HKDC1, see below 2001), and HKIV/GCK (Stoffel et al., 1992; Tanizawa et al., Section 3.4), nor there activity, thus it is possible that they may 1992) genes were found on 10q22, 2p12, 5q35, be expressed at low levels or at restricted sites thus escaping and 7p13 respectively (Table 1). detection using enzymatic assays. Alternatively, some of the In addition to the four known human hexokinase genes, the genes may have lost or changed enzymatic activity. previously identified hexokinase II pseudogene (Malkki et al., 1994; Ardehali et al., 1995), which maps to chromosome Xq21, 3.2. Hexokinase gene order is conserved among vertebrates was also found. The human HKII pseudogene has no introns, suggesting that it was derived from an mRNA transcript To determine whether hexokinases genes are conserved in (Malkki et al., 1994; Ardehali et al., 1995), and contains other vertebrate genomes, we searched the near complete internal stop codons and an insertion that would generate a genomes that are available from diverse vertebrate species for translational frameshift that should prevent the production of a hexokinase-like genes (Table 2, Figs. 1–4). Searches of functional protein (Fig. S1). Divergence of the human HKII mammalian genomes, including, rhesus macaque (M. mulatta), gene and pseudogene sequences suggested that the pseudogene mouse (Mus domesticus), rat (Rattus norvegicus), dog (Cannis originated about 14–16 million years ago (Ardehali et al., familiaris), cow (Bos taurus) and opossum (Monodelphus 1995). To confirm this date of origin for the HKII pseudogene domesticus) resulted in the identification of 5 hexokinase-like we searched the chimpanzee (Pan troglodytes) and rhesus genes in all of the species (Table 2). Initially several genes, macaque (Macca mullata) genomes, which diverged about 5 including HKI from the rat and HKDC1 from rat, dog, and cow, and 25 million years ago respectively from the human lineage were not found as annotated genes in the Ensembl genome (Kumar and Hedges, 1998), for a HKII pseudogene sequence. assemblies. Searches of the Ensembl genome assemblies for An X chromosome-linked HKII-like processed pseudogene sequences that potential could encode the missing genes sequence was identified in the chimpanzee but not the rhesus identified sequences similar to HKDC1 in dog and cow, although they had not been annotated as being a gene (see Table 2). No sequences most similar to HKI or HKDC1 were found in Table 1 the Ensembl rat genome assembly. A gene for rat HK1 likely Hexokinase-like genes in the human genome exists as a cDNA for this gene has been characterized a b Gene name Chromosomal location Accession number (accessions: NM_012734 for rat HKI, Schwab and Wilson, HK1 10q22.1 ENSG00000156515 1988). To complement our Ensemble database searches, we also HK2 2p12 ENSG00000159399 searched the genome assemblies maintained by NCBI. A rat ψ HK2 Xq21.1 AL412504.9 HKI gene was found within the NCBI genome assembly, and a HK3 5q25.2 ENSG00000160883 GCK 7p13 ENSG00000106633 sequence similar to HKDC1 was found adjacent to the rat HKI HKDC1 10q22.1 ENSG00000156510 gene, but this sequence had not been annotated or predicted to a Location identified within Ensembl genome assembly. be a gene (Table 2). In contrast to the Ensembl assemblies, the b Embl Gene ID for the protein coding genes. For ψHK2, NCBI accession NCBI dog and cow genome assemblies both predict genes number of genomic sequence. similar to HKDC1 (Table 2). 中国科技论文在线 http://www.paper.edu.cn D.M. Irwin, H. Tan / Comparative Biochemistry and Physiology, Part D 3 (2008) 96–107 99

Fig. 1. Synteny of HKI and HKDC1 genes. The relative orientation of genes adjacent to HKI and HKDC1 in diverse vertebrate genomes is shown. Human gene names are shown. Gene maps are from the Ensembl genome assemblies, with homologs to human genes shown. Species are human (Homo sapiens), mouse (Mus musculus), opossum (Monodelphus demnesticus), chicken (Gallus gallus), Xenopus (Xenopus tropicalis), zebrafish (Danio rerio) and medaka (Oryzias latipes). The chromosomal location of the human gene is indicated, with the homologous chromosome, or scaffold/contig for genomes without chromosome maps shown beside the species name. For zebrafish, the position on chromosome 13 of the duplicated genomic segment is indicated. Arrows show the direction of transcription of genes. Gene sizes and distance between genes are not to scale.

To aid in confirming the orthology of hexokinase genes we assemblies are still incomplete, and indeed differences exist examined the conservation of the gene order, or synteny, near between the Ensembl and NCBI genome assemblies for the each of the hexokinase genes. Not only are all five hexokinase- same species (e.g., rat). Therefore, some of the differences that like genes found in all mammalian species examined, but their we observed in gene order (e.g., cow HKI and HKDC1) may genomic arrangement in the genome is similar in most reflect incorrect genome assemblies rather than changes in mammals (Figs. 1–4). HK1 and HKDC1 were always found genome structure. together in tandem (Fig. 1), except in the cow where the two We also searched non-mammalian vertebrate genomes for genes were separated but still on the same chromosome. The hexokinase-like genes. While 5 hexokinase-like gene sequences SUPV3L1 and VPS26A genes were found 5′ to the HKI and were found in the chicken (Gallus gallus) genome, only three of HKDC1 genes, while most often the TSPAN15 gene was found the genes, HKI, HKII, and HKDC1, predict intact genes (Table 3′ to the hexokinases (Fig. 1). The HKII was found to have the 2, Figs. 1–4). The HKI and HKDC1 genes were in a genomic POLE4 and TACR1 gene located 3′ to the gene and SEMA4F arrangement similar to that seen in mammals (Fig. 1). While the located 5′ in placental mammals, although the FIGLA gene HKII gene was intact, the genomic neighbors of chicken HKII replaced SEMA4F in the genome of the marsupial mammal, the could not be determined as this gene sequence is on a DNA opossum (Fig. 2). Gene order around the HKIII gene and GCK fragment that is currently unassigned to the genome, and the genes were completely conserved in mammals (Figs. 3 and 4). genomic fragment does not contain any other genes (Fig. 2). The HKIII gene is flanked by the UIMC1 and UNC5A genes Previously, it had been reported that chicken liver was deficient while GCK is flanked by the YKT6 and MYL7 genes. These in glucokinase (Ureta et al., 1973; Cárdenas et al., 1998). A results indicate that all five genes existed in the common more recent paper, though, reports a near full-length GCK ancestor of mammals. It should be noted that most genome cDNA (accession AF525739) and identified an immunoreactive 中国科技论文在线 http://www.paper.edu.cn 100 D.M. Irwin, H. Tan / Comparative Biochemistry and Physiology, Part D 3 (2008) 96–107

Table 2 Hexokinase genes from diverse vertebrate species a Species HK1 HKDC1 HK2 HK3 GCK Homo sapiens ENSG00000156515 ENSG00000156510 ENSG00000159399 ENSG00000160883 ENSG00000106633 Macaca mulatta ENSMMUG00000015302 ENSMMUG00000003849 ENSMMUG00000011845 ENSMMUG00000002653 ENSMMUG00000002427 Mus musculus ENSMUSG00000037012 ENSMUSG00000020080 ENSMUSG000000006 ENSMUSG00000025877 ENSMUSG00000041798 Rattus norvegicus NP_036866 b Unannotated c ENSRNOG00000006116 ENSRNOG00000026235 ENSRNOG00000014447 Canis familiaris ENSCAFG00000013917 XP_546137 b ENSCAFG00000008265 ENSCAFG00000016584 ENSCAFG00000002944 Bos taurus ENSBTAG00000012380 LOC614824 b ENSBTAG00000013108 ENSBTAG00000014898 ENSBTAG00000032288 Monodelphus ENSMODG00000010645 ENSMODG00000010681 ENSMODG00000011328 ENSMODG00000005000 ENSMODG00000016167 domestica Gallus gallus ENSGALG00000004222 ENSGALG00000021039 ENSGALG0000000975 –– Xenopus tropicalis ENSXETG00000008860 ENSXETG00000008859 ENSXETG00000008520 ENSXETG00000019639 ENSXETG00000019003 Danio rerio ENSDARG00000039452 ENSDARG00000039451 ENSDARG00000026964 – ENSDARESTG00000016841 ENSDARG00000038702 ENSDARG00000038703 Oryzias latipes ENSORLG00000010420 ENSORLG00000010258 ENSORLG00000015439 ENSORLG00000019565 ENSORLG00000002010 ENSORLG00000000069 Takifugu rubripes SINFRUG00000125463 – SINFRUG00000162397 SINFRUG00000128934 SINFRUG00000145855 SINFRUG00000146976 Tetraodon GSTENG00025792001 GSTENG00025793001 GSTENG00031116001 GSTENG00014575001 GSTENG00029591001 nigroviridis GSTENG00004899001 Gasterosteus ENSGACG00000016084 ENSGACG00000016082 ENSGACG00000013109 ENSGACG00000020803 ENSGACG00000004420 aculeatus ENSGACG00000019782 a Ensemb gene IDs are listed. b NCBI protein accession numbers for sequences not found in Ensembl databases. c unannotated—gene is not known or predicted. protein as well as enzymatic activity (Berradi et al., 2005). Our previously reported partial cDNA (Fig. 4). Whether this searches of the genomic databases identified only a partial GCK represents an incorrect genome assembly or a mutated allele gene sequence that cannot encode a full-length protein, nor the of the gene needs further study. Previous studies had also

Fig. 2. Synteny of HKII genes. The relative orientation of genes adjacent to HKII in diverse vertebrate genomes is shown. Chromosomes and genes are organized as shown in Fig. 1. 中国科技论文在线 http://www.paper.edu.cn D.M. Irwin, H. Tan / Comparative Biochemistry and Physiology, Part D 3 (2008) 96–107 101

Fig. 3. Synteny of HKIII genes. The relative orientation of genes adjacent to HKIII in diverse vertebrate genomes is shown. Chromosomes and genes are organized as shown in Fig. 1.

suggested that the chicken lacked HKIII (Ureta et al., 1973). encode a functional and therefore should be a Consistent with that report, our genomic search was only able to pseudogene. In contrast to chicken, all five hexokinase-like find a partial HKIII-like gene sequence, in a genomic genes in the frog Xenopus tropicalis were found to be intact and environment similar to mammalian HKIII genes, in the chicken in genomic neighborhoods similar to those seen in mammals genome (Fig. 3). Our predicted chicken HKIII gene cannot (Table 2, Figs. 1–4).

Fig. 4. Synteny of GCK genes. The relative orientation of genes adjacent to GCK in diverse vertebrate genomes is shown. Chromosomes and genes are organized as shown in Fig. 1. 中国科技论文在线 http://www.paper.edu.cn 102 D.M. Irwin, H. Tan / Comparative Biochemistry and Physiology, Part D 3 (2008) 96–107

A larger number of hexokinase-like genes were found in fish that are about half the size of the other HKDC1 proteins (Fig. compared to other classes of vertebrates. A total of six S3). For each of these genes, a second hexokinase domain-like hexokinase-like genes were identified in all but one of the sequence could not found near the predicted HKDC1 genes. fish species that we examined that have near complete genomes Similarly, all of the fish HKIII sequences were predicted to have (Medaka, Oryzias latipes; Tetraodon, Tetraodon nigroviridis; only a single hexokinase domain (Fig. S5), and again a second Stickleback, Gasterosteus aculeatus; and Zebrafish, Danio hexokinase domain-like sequence was not found in genomic rerio)(Table 2, Figs. 1–4). The pufferfish Fugu (Takifugu sequences adjacent to these genes. These observations suggest rubripes ) was found to have only five hexokinase-like genes that in fish, all HKIII genes and some HKDC1 genes have (Table 2). Despite similar numbers of hexokinase-like genes, become truncated due to the loss of a hexokinase domain. the gene content differs between species. All five fish species were found to have two HKI genes, one HKII gene, and one 3.4. Is HKDC1 a hexokinase? GCK gene (Table 2, Figs. 1, 2, and 4). The number of HKDC1 genes varied between species, with D. rerio possessing 2 genes, Our genomic searches resulted in the identification of not T. rubripes having none, and other fish having a single gene only the previously well-characterized HKI, HKII, HKIII, and (Table 2, Fig. 1). The HKIII gene was not found in D. rerio, but GCK genes (Wilson, 1995; Cárdenas et al., 1998) but also of a was identified in the remaining four species (Table 2, Fig. 3). previously unknown hexokinase-like gene HKDC1. HKDC1 All of the fish hexokinase-like genes were found in genomic genes were found to be present in all of the genomes we neighborhoods that resembled the mammalian genes, suggest- examined, indicating that HKDC1 is a conserved gene, and ing that they are orthologous, although some rearrangements therefore is likely to be functional. If HKDC1 is functional, then have occurred (Figs. 1–4). it must be expressed. To determine whether human HKDC1 is The conservation of synteny near each of the hexokinase expressed, we searched the human EST database, resulting in genes suggests that we have identified orthologous genes the identification of more than 100 EST for this gene. The throughout vertebrates, and that gene content and gene order cataloguing of ESTs by Unigene (www.ncbi.nlm.nih.gov/ have been conserved since the fish–mammal divergence. The UniGene/) suggests that human HKDC1 is widely expressed, presence of additional genes in fish suggests that some with highest levels of expression observed in pharynx, , duplication events have occurred on this lineage. Since all colon, esophagus, and eye. We searched EST databases for five of the hexokinase-like genes are present in the genomes of other species including mouse (Mus musculus) and zebrafish species of all classes of vertebrates that we examined (D. rerio). Fewer ESTs were found in the mouse, with highest (mammals, birds, fogs, and bony fish), this suggests that the levels of expression found in eye and extraembryonic tissue. In hexokinase gene family originated prior to the bony fish- zebrafish, an even smaller number of ESTs were identified for mammal divergence. the duplicated HKDC1 genes. The two zebrafish genes appeared to have distinct expression patterns with one gene 3.3. Structure of vertebrate hexokinases expressed in the digestive tract and other in gills. EST database searches demonstrate that HKDC1 is an expressed gene, We have identified a large number of hexokinase genes from therefore potentially function, and like many other hexokinase a diverse collection of vertebrate species (Table 2). To genes has diverse sites of expression. complement these sequences we also searched the NCBI Despite extensive searches for proteins with hexokinase database for additional vertebrate hexokinase and glucokinase enzymatic activities, only four different hexokinase have been protein sequences (Table S1). These searches resulted in the identified in diverse vertebrates, and these are explained by identification of hexokinase and glucokinase protein sequences HKI, HKII, HKIII, and GCK (Wilson, 1995, Cárdenas et al., from a few additional mammalian, frog, and fish species. To 1998). These observations would tend to suggest that HKDC1 determine whether changes in the structure of these proteins does not have hexokinase activity. The recent characterization have occurred within vertebrates, we aligned each of the of an ADP-dependent glucokinase in mammalian genomes orthologous groups of sequences (Figs. S2–S6). The alignments (Ronimus and Morgan, 2004) though, does raise the possibility indicate that the HKI, HKII, and GCK proteins have been that some hexokinase activities may have been missed by these largely conserved in structure (Figs. S2, S4, and S6), and searches. Amino acid residues necessary for glucose and ATP differences that were observed for these proteins most likely binding in glucokinase have been identified by molecular represent incorrect gene prediction resulting in loss of N- or C- modeling and site-directed mutagenesis (Xu et al., 1994, 1995; terminal sequences (e.g., P. troglodytes HKI N-terminus, D. Marotta et al., 2005), and the homologous positions in human rerio HKII N-terminus, and D. rerio GCK C-terminus) or the hexokinases are shown in Fig. S1. The predicted human inclusion of introns within the predicted open reading frame HKDC1 amino acid sequence possesses conserved amino acid (e.g., T. nigroviridis HKIA). While similar variation, likely due residues thought to be necessary for glucose-binding in both the to incorrect prediction, was also observed in the HKDC1 and N- and C-terminal domains (residues 208, 209, 235, 260, 294, HKIII sequences (e.g., R. norvegicus HKDC1 and T. rubripes 655, 656, 682, 707, and 741, Fig. S1) as well as ATP-binding HKIII), significant structural changes were also observed (Figs. sites in the C-terminal domain (residues 532, 536,678, 679, and S3 and S5). For HKDC1, three of the fish genes predict a 787, Fig. S1). Conservation of the glucose and ATP-binding protein with only a single hexokinase domain, yielding proteins sites raises the potential that the C-terminal domain of HKDC1 中国科技论文在线 http://www.paper.edu.cn D.M. Irwin, H. Tan / Comparative Biochemistry and Physiology, Part D 3 (2008) 96–107 103

potentially has hexokinase activity. Further biochemical should yield genes on different chromosomes, as observed in characterization of HKDC1 is necessary to confirm whether O. latipes (Fig. 1) and other fish (data not shown), but the two this protein has hexokinase enzymatic activity. D. rerio HKI genes are also located on the same chromosome, which strengthens the suggestion that they are products of a 3.5. Hexokinases in Ciona and sea urchin different gene duplication event. This would also mean that D. rerio has lost one of the pair of duplicated HKI genes from Previous attempts to resolve the evolutionary origin of the the genome duplication event. A duplication event that occurred vertebrate hexokinases have used yeast hexokinase and separately on the D. rerio lineage could also explain why this glucokinase as the outgroups to root their trees (Griffin et al., species also has two HKDC1 genes (Fig. 1). The two D. rerio 1991; Cárdenas et al., 1998). The extremely divergent yeast HKDC1 genes, like the HKI genes, are more closely related to sequences provide little ability to revolve divergences that each other than to any other HKDC1 gene (see Fig. S9). Since occurred near the origin of vertebrates. The use of more closely the HKI and HKDC1 genes are in tandem, a single genomic related sequences should better resolve divergence events that duplication event could have duplicated both genes, as well as occurred near the origin of vertebrates. We searched the genome other adjace genes (see Fig. 1) on the D. rerio lineage. sequences from two species of urochrodates, C. intestinalis and Apart from the duplicated HKDC1 genes in D. rerio, the C. savignyi (both sea squirts) available at Ensembl and an remainder of the HKDC1 phylogeny is as expected (Fig. S9). echinoderm, S. purpuratus (sea urchin) at NCBI, for hexoki- Similarly, for HKII and HKIII the biological relationships are nase-like sequences. Urochrodates and echinoderms are rela- largely reflected by the sequence phylogenies (Figs. S10 and tively closely related to vertebrates, but are thought to have S11). For all three of these genes, only a single gene was found diverged prior to the large scale gene duplications that occurred in fish (except for the duplicated HKDC1 gene in D. rerio) on the vertebrate lineage (Hedges, 2002). Our searches (Table 2, Figs. 1–3), suggesting that if these genes were identified three hexokinase-like genes in each of C. savignyi duplicated during a fish-specific whole genome duplication, one and S. purpuratus and two hexokinase-like genes in C. of the two gene products was lost before the radiation of fish. intestinalis (Table S1, Fig. S6). All seven of these hexokinase- The phylogeny of fish species suggested by the GCK genes, like genes are predicted to encode a single hexokinase domain though, differs from the species phylogeny suggested by the (Fig. S6) thus should be similar in size to the hexokinase that has other hexokinase genes (compare Fig. S12 to Figs. S8–S11). been previously purified from an echinoderm, the starfish Most hexokinase genes indicate that the two pufferfish species (Asterias amurensis)(Mochizuki and Hori, 1977). (T. rubripes and T. nigroviridis) are deep within the fish phylogeny (Figs. 8–11), while in the GCK phylogeny the 3.6. Origin of vertebrate hexokinase genes pufferfish are the earliest diverging fish lineage (Fig. S12). A possible explanation for the conflicting phylogenies could be Genomic synteny mapping (Figs. 1–4) showed that the HKI, that the pufferfish GCK genes are paralogous to the GCK genes HKDC1, HKII, HKIII, and GCK genes are present in a diverse in the remaining fish species. The GCK genes may be para- array of vertebrate species, indicating that these genes diverged logous if differential loss of duplicated GCK genes, which were from each other prior to the divergence of fish and mammals. To generated by the fish-specific whole genome duplication, determine the relationships between the vertebrate hexokinase occurred on different fish lineages. we conducted phylogenetic analysis using both distance GCK has only a single hexokinase domain in contrast to the (neighbor-joining with JTT and PAM distances) and parsimony remaining vertebrate hexokinase genes (Wilson, 1995; Cárde- methods. For all analyses similar phylogenetic results were nas et al., 1998). To determine the relationships of GCK to the obtained. First we conducted an analysis of each gene separately larger hexokinases, and within the family of hexokinases we (Figs. S8–S12). In general, the phylogenies that were generated established a phylogeny of the hexokinase domains from all of for each of the five vertebrate hexokinase genes reflect the these genes (Figs. 5 and S13). Our phylogeny would not only expected biological relationships, supporting our conclusion show the relationships between the members of this gene that they are orthologous sequences. A few unexpected family, but would also place the origin of the duplication of the phylogenetic relationships, though, were revealed by our gene hexokinase domain. We used hexokinases from closely related phylogenies. non-vertebrate species to root our tree. In our phylogeny we A pair of HKI genes was found in all fish species (Table 2), found that the hexokinase domain sequences for the three which suggests that a duplication of this gene occurred in the groups, echinoderms, urochrodates, and vertebrates, were ancestor of fish. The HKI duplication may be part of the fish- monophyletic (Figs. 5 and S13), implying that the multiple specific genome duplication that occurred in the ancestor of hexokinase genes found in species in each of these group are the teleost fish (Meyer and Van de Peer, 2005). While most of the products of gene duplications events that occurred indepen- fish HKI genes yield a phylogeny consistent with retention dently on each of these lineage. Since both the echinoderm and of genes that duplicated prior to the fish radiation, the pair of urochordate hexokinase genes, like other non-vertebrate D. rerio HKI genes are more closely related each other than to hexokinases (Wilson, 1995; Cárdenas et al., 1998), contain any other gene (Fig. S8). This phylogeny suggests that the two only a single hexokinase domain, this strongly supports the D. rerio HKI genes are the products of a much more recent gene conclusion that the ancestral vertebrate hexokinase gene duplication event. Duplication of HKI by genome duplication contained only a single hexokinase domain. Our phylogeny 中国科技论文在线 http://www.paper.edu.cn 104 D.M. Irwin, H. Tan / Comparative Biochemistry and Physiology, Part D 3 (2008) 96–107

of glucokinase within vertebrate hexokinases has been debated (Wilson, 1995; Cárdenas et al., 1998). Griffin et al. (1991) concluded that GCK diverged from other vertebrate hexoki- nases after duplication of the hexokinase domain, and GCK subsequently lost its N-terminal hexokinase domain. In contrast, Cárdenas et al. (1998) conclude that GCK diverged from the vertebrate hexokinases before the duplication of the hexokinase domain, thus did not have to hypothesize the deletion of a hexokinase domain. Both of these analysis, though, relied on a very small number of vertebrate hexokinase and glucokinase sequences, and used the very distant yeast sequences to root their analyses (Griffin et al., 1991; Cárdenas et al., 1998). Our new analysis with a large number of diverse vertebrate hexokinase-like sequences as well as hexokinases from close relative of vertebrates to root our analysis suggests that glucokinase is most closely related to the C-terminal hexokinase domains of vertebrate hexokinases and diverged from the hexokinases after duplication of the hexokinase domain (Figs. 5 and S13), a phylogeny similar to that proposed by Griffin et al. (1991). Given our greater number of sequences, and better outgroups, we have greater confidence in our phylogeny. The hexokinase genes duplicated and diverged from each other very early in vertebrate evolution, at about the time when two genome duplications have been hypothesized to occur (Sidow, 1996). Duplication of hexokinase genes through genome duplications could explain their existence in all vertebrate species examined and their distribution on to four different chromosomes (Table 1, Figs. 1–4). Genome duplica- tions, though, cannot explain the location of the HKI and HKDC1 genes. The HKI and HKDC1 gene sequences are most closely related to each other (Figs. 5 and S13), thus diverged after the radiation of the other hexokinases, and are found adjacent to each other in genomes (Fig. 1). These relationships Fig. 5. Phylogeny of vertebrate hexokinase domains. Summary of the phylogeny suggest that the HKI and HKDC1 genes are the product of a of hexokinase domains from HKI, HKII, HKIII, HKDC1, and GCK genes from tandem gene duplication event that occurred after the genome diverse vertebrates rooted using echinoderm (sea urchin) are urochrordate duplication events. (Ciona — referring to Ciona intestinalis and Ciona savignyi) sequences. The full phylogenetic tree is shown in Fig. S13. Triangles at tips of branches refer to the diversity of hexokinase domain sequences that make up each branch. Solid 3.7. Evolution of vertebrate hexokinase genes diamond indicates the position of the inferred hexokinase domain duplication event in the ancestor of vertebrate hexokinases. The C-terminal hexokinase Our phylogeny allows us to develop a model for the domains of the vertebrate hexokinases are in the upper part of the tree while the evolution of the vertebrate hexokinase gene family (Fig. 6). N-terminal hexokinase domains are in the lower part. Tandem duplication of a gene containing a single hexokinase domain yielded a gene containing two hexokinase domains very indicates that the duplication of the hexokinase domain, to yield early in vertebrate evolution, but after divergence of vertebrates the 100 kDa hexokinases, occurred after the urochordate– from urochordates. The initial domain duplication event should vertebrate divergence (Figs. 5 and S13). After duplication of the have generated a hexokinase with two active domains. If the hexokinase domain, the five vertebrate hexokinases diverged domain duplication event preceded the two whole genome from each other (Figs. 5 and S13). For both the N- and C- duplication that also occurred in the early vertebrate (Sidow, terminal hexokinase domain, identical phylogenies for HKI, 1996), then the genome duplication events could explain the HKII, HKIII, and HKDC1 are inferred, where HKI and HKDC1 earliest divergences of the genes within this gene family (Fig. are most closely related, with HKII being more closely related 6). A later gene duplication event involving one of the four to HKI/HKDC1 than HKIII (Figs. 5 and S13). The relationships genes generated by the two genome duplications then generated inferred for HKI, HKII, and HKIII are similar to those proposed the tandemly linked HKI and HKDC1 genes (Fig. 1). Changes earlier based on a smaller number of sequences (Cárdenas et al., to both gene structure and enzymatic activity have occurred 1998). subsequent to the gene duplication events. A more intriguing question concerns the relationship of GCK has only a single hexokinase domain, yet our hypo- glucokinase to the other hexokinases. The phylogenetic position thesis indicates that it had an ancestor that had two hexokinase 中国科技论文在线 http://www.paper.edu.cn D.M. Irwin, H. Tan / Comparative Biochemistry and Physiology, Part D 3 (2008) 96–107 105

Fig. 6. Evolution of vertebrate hexokinase genes. Proposed model for the evolution of the vertebrate hexokinase gene family. The boxes indicate hexokinase domains, with N and C referring to N-terminal and C-terminal domains respectively. XN and NX indicate inactive N-terminal hexokinase domains, with question marks (?) indicating uncertain status. An initial duplication and fusion of a single hexokinase domain-containing gene yield a hexokinase that contains two hexokinase domains. Gene duplications, likely through genome duplications yielded four hexokinase genes on different chromosomes. A tandem gene duplication generated the HKI and HKDC1 genes. Although GCK had an ancestor that had two hexokinase domains, it has now lost its N-terminal hexokinase domain. Independent mutations have inactivated the hexokinase activity of the N-terminal hexokinase domains of HKI and HKIII. As hexokinase activity has not been described for HKDC1, there is uncertainty in the timing and number of enzymatic mutations that have occurred to HKI and HKDC1.

domains (Fig. 6). Our phylogenetic analysis (Figs. 5 and S13) been noted that the N-terminal domains of mammalian indicates that the hexokinase domain in GCK is most closely hexokinases are evolving rapidly than their C-terminal domains related to the C-terminal hexokinase domains of other (Cárdenas et al., 1998), and the N-terminal domains lack hexokinases, suggesting that the N-terminal domain of the hexokinase activity (Tsai and Wilson, 1997; Tsai, 1999). Since ancestral GCK was lost. The simplest hypothesis for the loss of fish HKIII only have a single domain, if these proteins do have the N-terminal domain is by a deletion event that was mediated enzymatic activity then this domain must have hexokinase by sequences, potentially repetitive DNA, in an intron located in active. Our phylogeny also shows that only mammalian HKIII N-terminal part of the hexokinase domains. As both hexokinase N-terminal domains are evolving rapidly, and that the N- domains have similar exon–intron gene structures, recombina- terminal domain of the frog X. tropicalis has accumulated a tion between homologous introns would result in maintenance of similar amount of change as fish HKIII sequences or other the open reading frame and the deletion of one of the two hexokinase domains (Fig. S13). These observations suggest that hexokinase domains. A recombination event involving an intron fish HKIII, and the N-terminal domain of X. tropicalis HKIII near the N-terminal end of the hexokinase domain would result may still retain enzymatic activity, and that only after the in a product that retains the genes promoter and a single mammals–amphibian divergence did the N-terminal hexoki- hexokinase domain that is mostly derived, and thus most related nase domain of HKIII lose activity, and with that show an to, the C-terminal hexokinase domain. Several other hexokinase acceleration in the rate of sequence evolution. genes appear to have lost one of their two domains, including The N-terminal domains of HKI have also lost hexokinase fish HKIII genes and some fish HKDC1 genes (Figs. S3 and S5). activity (White and Wilson, 1989; Tsai, 1999). Since our In each of these cases it appears that the C-terminal domain that phylogenetic analysis suggests that the mutation that caused the has been lost, a simpler event that only requires is a mutation loss of activity in the N-terminal hexokinase domain of HKIII (e.g., point mutation, insertion, or deletion) to occur downstream only occurred after the mammal–amphibian divergence, and of the N-terminal hexokinase domain. since HKI and HKII share a more recent common ancestor than Surprisingly, our phylogeny indicates that the hexokinase HKI and HKIII, and the N-terminal domain of HKII has domains of the fish HKIII genes are more closely related to the enzymatic activity (Tsai and Wilson, 1996), the mutation that N-terminal domains rather than C-terminal domains of mam- caused the loss of activity in the N-terminal domain of HKI malian HKIII genes (Fig. S13). In contrast, Clustal W aligned must be independent of that in HKIII (see Fig. 6). Sequence the fish HKIII hexokinase domain sequences to the C-terminal analysis of the N-terminal hexokinase of HKDC1 suggests domain of mammalian HKIII genes (Fig. S5). Previously it had that it may not have hexokinase activity (see Fig. S1), thus 中国科技论文在线 http://www.paper.edu.cn 106 D.M. Irwin, H. Tan / Comparative Biochemistry and Physiology, Part D 3 (2008) 96–107 potentially HKI and HKDC1 share a common mutation causing Griffin, L.D., Gelb, B.D., Adams, V., McCabe, E.R., 1992. Developmental the loss of activity in their N-terminal domains (Fig. 6). expression of hexokinase 1 in the rat. Biochim. Biophys. Acta 1129, 309–317. Alternatively, the N-terminal domain of HKDC1 may have lost Hedges, S.B., 2002. The origin and evolution of model organisms. Nat. Rev., hexokinase activity due to an independent mutation (Fig. 6). In Genet. 3, 838–849. addition, we do not know whether additional inactivating Kamata, K., Mitsuya, M., Nishimura, T., Eiki, J., Nagata, Y., 2004. Structural mutations have occurred in the C-terminal hexokinase domain basis for allosteric regulation of the monomeric allosteric enzyme human – of HKDC1. glucokinase. Structure 12, 429 438. Katzen, H.M., Schimke, R.T., 1965. Multiple forms of hexokinase in the rat: tissue distribution, age dependency, and properties. Proc. Natl. Acad. Sci. 4. Conclusions U. S. A. 54, 1218–1225. Kogure, K., Shinohara, Y., Terada, H., 1993. Evolution of the type II hexokinase Our genomic searches have identified a novel hexokinase- gene by duplication and fusion of the glucokinase gene with conservation of – like gene, HKDC1. HKDC1 is expressed and its sequence has its organization. J. Biol. Chem. 268, 842 8424. Kumar, S., Hedges, S.B., 1998. A molecular timescale for vertebrate evolution. been conserved in diverse vertebrates. Since HKDC1 is Nature 392, 917–920. conserved, and retains amino acid residues responsible for Mahalingam, B., Cuesta-Munoz, A., Davis, E.A., Matschinsky, F.M., Harrison, hexokinase activity, it is possible that HKDC1 is a hexokinase. R.W., Weber, I.T., 1999. Structural model of human glucokinase in complex Phylogenetic analysis shows that HKDC1 is most closely with glucose and ATP. Diabetes 48, 1698–1705. related to HKI. In addition our phylogenetic analysis resolves Malkki, M., Laakso, M., Deeb, S.S., 1994. Structure of the human hexokinase II gene. Biochem. Biophys. Res. Commun. 205, 490–496. the relationships among vertebrate hexokinase genes, and Marotta,D.E.,Anand,G.R.,Anderson,T.A.,Miller,S.P.,Okar,D.A.,Levitt,F.G., demonstrates that GCK is descendent from a hexokinase that Lange, A.J., 2005. Identification and characterization of the ATP-binding site in had two hexokinase domains. human pancreatic glucokinase. Arch. Biochem. Biophys. 436, 23–31. 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