Binding of non-catalytic ATP to human hexokinase I highlights the structural components for enzyme–membrane association control

Rosano, Camillo; Sabini, Elisabetta; Rizzi, Menico; Deriu, Daniela; Murshudov, Garib N.; Bianchi, Marzia; Serafini, Giordano; Magnani, Mauro; Bolognesi, Martino

doi:10.1016/s0969-2126(00)80032-5

Cited by 51 publications

(51 citation statements)

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“…More specifically it belongs to the "actin-like ATPase domain" superfamily of proteins, often referred to as the ASKHA (acetate and sugar kinase/hsp70/actin) superfamily (8,23). Proteins in this superfamily include the ATPase domain of actin (22,29) and hsp70 (19), acetate kinase (11), several sugar kinases, such as hexokinase (44,48), glycerol kinase (24), ATP-and ADP-dependent glucokinases (25,35), as well as 2-hydroxyglutaryl-CoA dehydratase component A (2-HG-CoA dehydratase Comp A) (33). Similar to all other members of the ASKHA superfamily, the PanK Tm monomer contains two domains that have the same fold and are considered to be a result of gene duplication (Fig.…”

Section: Resultsmentioning

confidence: 99%

Crystal Structure of a Type III Pantothenate Kinase: Insight into the Mechanism of an Essential Coenzyme A Biosynthetic Enzyme Universally Distributed in Bacteria

et al. 2006

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Pantothenate kinase (PanK) catalyzes the first step in the five-step universal pathway of coenzyme A (CoA) biosynthesis, a key transformation that generally also regulates the intracellular concentration of CoA through feedback inhibition. A novel PanK protein encoded by the gene coaX was recently identified that is distinct from the previously characterized type I PanK (exemplified by the Escherichia coli coaA-encoded PanK protein) and type II eukaryotic PanKs and is not inhibited by CoA or its thioesters. This type III PanK, or PanK-III, is widely distributed in the bacterial kingdom and accounts for the only known PanK in many pathogenic species, such as Helicobacter pylori, Bordetella pertussis, and Pseudomonas aeruginosa. Here we report the first crystal structure of a type III PanK, the enzyme from Thermotoga maritima (PanK Tm ), solved at 2.0-Å resolution. The structure of PanK Tm reveals that type III PanKs belong to the acetate and sugar kinase/heat shock protein 70/actin (ASKHA) protein superfamily and that they retain the highly conserved active site motifs common to all members of this superfamily. Comparative structural analysis of the PanK Tm active site configuration and mutagenesis of three highly conserved active site aspartates identify these residues as critical for PanK-III catalysis. Furthermore, the analysis also provides an explanation for the lack of CoA feedback inhibition by the enzyme. Since PanK-III adopts a different structural fold from that of the E. coli PanK-which is a member of the "P-loop kinase"superfamily-this finding represents yet another example of convergent evolution of the same biological function from a different protein ancestor.Pantothenate kinase (PanK; EC 2.7.1.33) catalyzes the ATPdependent phosphorylation of pantothenate (vitamin B 5 ) to give 4Ј-phosphopantothenate. This reaction represents the first and committed step in the universal biosynthetic pathway of coenzyme A (CoA) (6, 27, 32). Because CoA is a ubiquitous and essential cofactor in all organisms, genes coding for the five enzymes that make up this pathway-including PanK-are essential for their survival and growth (6,27).Three distinct types of PanK, as differentiated by primary sequence analysis and kinetic properties, have been characterized so far. Type I PanKs (PanK-I) are found exclusively in eubacterial species and are exemplified by the Escherichia coli enzyme encoded by the coaA gene (46, 47). The second type of PanK (PanK-II) is found mainly in eukaryotes, including yeast and various fungi, plants, and mammals (12, 41-43). Interestingly, PanKs from a few gram-positive bacteria, such as Staphylococcus aureus (PanK Sa ) and several bacilli, are also included in this group based on sequence homology, although the bacterial enzymes exhibit certain catalytic characteristics different from their eukaryotic counterparts (15,31). Recently, a third type of PanK (PanK-III) was identified which represents the only known pantothenate kinase activity in many pathogenic bacteria, including Helicobacter pylor...

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Section: Resultsmentioning

confidence: 99%

Crystal Structure of a Type III Pantothenate Kinase: Insight into the Mechanism of an Essential Coenzyme A Biosynthetic Enzyme Universally Distributed in Bacteria

et al. 2006

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“…A search for similar structures by using the DALI server (29; http://www.ebi.ac.uk/dali/) found that the most similar structures were human brain hexokinase I (PDB code 1QHA) (57) and hexokinase B (also known as hexokinase PII) from S. cerevisiae (PDB 2YHX) (4). Yeast hexokinase PII is somewhat larger than ecGlK, comprising 486 residues (38), while human hexokinase type I is much larger, consisting of two ϳ50-kDa chains (72).…”

Section: Resultsmentioning

confidence: 99%

“…Several crystal structures of hexokinases have been determined, including hexokinase A(PI) (10) and B(PII) (4,38), both from Saccharomyces cerevisiae, rat type I and Schistosoma mansoni hexokinase (46), human brain type I (1,2,3,57), and, recently, human type IV (glucokinase) (36). Only three microbial ADP-dependent GlK structures are available, all from sequence group I.…”

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Crystal Structures ofEscherichia coliATP-Dependent Glucokinase and Its Complex with Glucose

Лунин

Schrag

et al. 2004

J Bacteriol

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their narrow specificity for glucose as a substrate. While structural information is available for ADP-dependent glucokinases from Archaea, no structural information exists for the large sequence family of eubacterial ATPdependent glucokinases. Here we report the first structure determination of a microbial ATP-dependent glucokinase, that from E. coli O157:H7. The crystal structure of E. coli glucokinase has been determined to a 2.3-Å resolution (apo form) and refined to final R work /R free factors of 0.200/0.271 and to 2.2-Å resolution (glucose complex) with final R work /R free factors of 0.193/0.265. E. coli GlK is a homodimer of 321 amino acid residues. Each monomer folds into two domains, a small ␣/␤ domain (residues 2 to 110 and 301 to 321) and a larger ␣؉␤ domain (residues 111 to 300). The active site is situated in a deep cleft between the two domains. E. coli GlK is structurally similar to Saccharomyces cerevisiae hexokinase and human brain hexokinase I but is distinct from the ADP-dependent GlKs. Bound glucose forms hydrogen bonds with the residues Asn99, Asp100, Glu157, His160, and Glu187, all of which, except His160, are structurally conserved in human hexokinase 1. Glucose binding results in a closure of the small domains, with a maximal C␣ shift of ϳ10 Å. A catalytic mechanism is proposed that is consistent with Asp100 functioning as the general base, abstracting a proton from the O6 hydroxyl of glucose, followed by nucleophilic attack at the ␥-phosphoryl group of ATP, yielding glucose-6-phosphate as the product.Growth of Escherichia coli by using various fermentable sugars as carbon sources, including glucose, maltose, galactose, and sucrose, primarily involves the phosphoenolpyruvate-dependent phosphotransferase system (PTS) (reviewed in reference 54). However, a secondary, PTS-independent system for utilization of glucose also exists, consisting of glucose uptake by galactose permease (GalP; galactose proton symporter), followed by phosphorylation by glucokinase (GlK; EC 2.7.1.2) to yield the metabolic intermediate glucose-6-phosphate. Although glk mutant strains of E. coli (43) and Bacillus subtilis (63) are not visibly physiologically impaired, this enzyme retains the important function of phosphorylating any free intracellular glucose. Free cytoplasmic glucose may arise from disaccharide hydrolysis, for example, the cleavage of trehalose phosphate in Bacillus subtilis (25), or from metabolism of maltose or isomaltose (61). Indeed, studies of a PTS Ϫ E. coli strain have shown that a growth rate approximately 89% that of wild-type cells can be obtained by overexpression of GalP alone, suggesting that glucose transport, not GlK-dependent phosphorylation, is limiting growth (28). There is considerable industrial interest in enhancing the ability of E. coli to transport and phosphorylate glucose in a PTS-independent manner due to the ability of these strains to direct more carbon flux to aromatic synthesis pathways (20,21,28).Microbial glucokinases can be divided into three families based on sequ...

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“…X-ray crystallographic structures of HKI are available (11)(12)(13)(14)(15). The wild-type enzymes are dimers of 100 kDa subunits (12), but an engineered mutant HKI crystallizes as a monomer, adopting a conformation similar to that of the subunit of the dimer (14).…”

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confidence: 99%

Glucose 6-Phosphate Release of Wild-type and Mutant Human Brain Hexokinases from Mitochondria

Skaff

Kim

Tsai

et al. 2005

Journal of Biological Chemistry

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One molecule of glucose 6-phosphate inhibits brain hexokinase (HKI) with high affinity by binding to either one of two sites located in distinct halves of the enzyme. In addition to potent inhibition, glucose 6-phosphate releases HKI from the outer leaflet of mitochondria; however, the site of glucose 6-phosphate association responsible for the release of HKI is unclear. The incorporation of a C-terminal polyhistidine tag on HKI facilitates the rapid purification of recombinant enzyme from Escherichia coli. The tagged construct has N-formyl methionine as its first residue and has mitochondrial association properties comparable with native brain hexokinases. Release of wild-type and mutant hexokinases from mitochondria by glucose 6-phosphate follow equilibrium models, which explain the release phenomenon as the repartitioning of ligand-bound HKI between solution and the membrane. Mutations that block the binding of glucose 6-phosphate to the C-terminal half of HKI have little or no effect on the glucose 6-phosphate release. In contrast, mutations that block glucose 6-phosphate binding to the N-terminal half require ϳ7-fold higher concentrations of glucose 6-phosphate for the release of HKI. Results here implicate a primary role for the glucose 6-phosphate binding site at the N-terminal half of HKI in the release mechanism.Brain tissue relies almost exclusively on glucose as its source of energy. Approximately 25% of circulating blood glucose and 20% of consumed oxygen is utilized by the brain, an organ that constitutes less than 2% of the human body mass (1). Although four isozymes of hexokinase (ATP:D-hexose 6-phosphotransferase (2.7

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Binding of non-catalytic ATP to human hexokinase I highlights the structural components for enzyme–membrane association control

Cited by 51 publications

References 50 publications

Crystal Structure of a Type III Pantothenate Kinase: Insight into the Mechanism of an Essential Coenzyme A Biosynthetic Enzyme Universally Distributed in Bacteria

Crystal Structure of a Type III Pantothenate Kinase: Insight into the Mechanism of an Essential Coenzyme A Biosynthetic Enzyme Universally Distributed in Bacteria

Crystal Structures ofEscherichia coliATP-Dependent Glucokinase and Its Complex with Glucose

Glucose 6-Phosphate Release of Wild-type and Mutant Human Brain Hexokinases from Mitochondria

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