Abstract Infection with HIV or SIV can elicit potent and broadly reactive neutralizing antibodies against HIV, as well as disease modifying CTL against SIV. In order to elicit similar responses by vaccination, we have developed live attenuated rubella viral vectors to express HIV and SIV antigens. Safety and immunogenicity of rubella vaccine is well known; it elicits mucosal immunity, and one dose protects for life (against rubella). We have identified two insertion sites, in the nonstructural (ns) and structural regions, where foreign genes can be expressed without affecting viral growth. We have made 15 vectors expressing HIV MPER and SIV Gag antigens containing multiple CTL epitopes. Inserts at the ns site were expressed as rubella early genes. Inserts at the structural site were over-expressed as late genes and processed with the structural polyprotein. Some of the structural inserts were incorporated into virions and displayed on their surface. The vector should be safe, since loss of the insert would revert to the vaccine strain. We are using rubella because it grows well in rhesus macaques, which provide a good model of immunogenicity and protection against SIV or SHIV challenge. In preliminary studies, one of the vectors is growing in vivo, and it has elicited antibodies to the vector. These vectors will be useful in determining the role of a live vector in eliciting protective immunity and for optimizing its use in a prime and boost strategy.
Abstract Background Live attenuated viruses are among our most potent and effective vaccines. For human immunodeficiency virus, however, a live attenuated strain could present substantial safety concerns. We have used the live attenuated rubella vaccine strain RA27/3 as a vector to express SIV and HIV vaccine antigens because its safety and immunogenicity have been demonstrated in millions of children. One dose protects for life against rubella infection. In previous studies, rubella vectors replicated to high titers in cell culture while stably expressing SIV and HIV antigens. Their viability in vivo , however, as well as immunogenicity and antibody persistence, were unknown. Results This paper reports the first successful trial of rubella vectors in rhesus macaques, in combination with DNA vaccines in a prime and boost strategy. The vectors grew robustly in vivo , and the protein inserts were highly immunogenic. Antibody titers elicited by the SIV Gag vector were greater than or equal to those elicited by natural SIV infection. The antibodies were long lasting, and they were boosted by a second dose of replication-competent rubella vectors given six months later, indicating the induction of memory B cells. Conclusions Rubella vectors can serve as a vaccine platform for safe delivery and expression of SIV and HIV antigens. By presenting these antigens in the context of an acute infection, at a high level and for a prolonged duration, these vectors can stimulate a strong and persistent immune response, including maturation of memory B cells. Rhesus macaques will provide an ideal animal model for demonstrating immunogenicity of novel vectors and protection against SIV or SHIV challenge.
Native hepatitis B surface antigen (HBsAg) spontaneously assembles into 22-nm subviral particles. The particles are lipoprotein micelles, in which HBsAg is believed to span the lipid layer four times. The first two transmembrane domains, TM1 and TM2, are required for particle assembly. We have probed the requirements for particle assembly by replacing the entire first or third TM domain of HBsAg with the transmembrane domain of HIV gp41. We found that either TM domain of HBsAg could be replaced, resulting in HBsAg-gp41 chimeras that formed particles efficiently. HBsAg formed particles even when both TM1 and TM3 were replaced with the gp41 domain. The results indicate remarkable flexibility in HBsAg particle formation and provide a novel way to express heterologous membrane proteins that are anchored to a lipid surface by their own membrane-spanning domain. The membrane-proximal exposed region (MPER) of gp41 is an important target of broadly reactive neutralizing antibodies against HIV-1, and HBsAg-MPER particles may provide a good platform for future vaccine development.
Abstract Live attenuated rubella virus has a number of desirable features for a live viral vector. It has a good safety profile, so it can immunize while growing exponentially in the host. It immunizes at a low dose, and one dose protects (against rubella) for life. It grows well in rhesus macaques, which allow a viral challenge. It has no DNA intermediate, cannot integrate, and usually does not persist in the host. A full length infectious cDNA clone of rubella was available for modification, but prior efforts to express foreign genes were frustrated by genetic instability and size limitations on the insert. A deletion of 507 bp in nonstructural gene P150 was permissive, so we asked whether this deletion could make room for insertion of zoanthus GFP (792 bp). By using the deletion/insertion strategy, zGFP was stably expressed for at least 12 passages. In the zGFP hybrid, rubella structural proteins were expressed at normal levels, while the vector grew to high titer (4 X 10^6/ml). Normal P150 function was detected by P150-GFP labeling of functional replication centers in living cells. Rubella may be useful for expressing vaccine antigens as large as most viral proteins, and it is stable enough for vaccine production followed by growth in vivo. This approach may extend the range and utility of rubella to include immunization against other viruses for which attenuation is not currently feasible.
ADP-l-glycero-d-mannoheptose 6-epimerase is a 240 kDa NAD-dependent nucleotide diphosphosugar epimerase from Escherichia coli K12 which catalyzes the interconversion of ADP-d-glycero-d-mannoheptose and ADP-l-glycero-d-mannoheptose. ADP-l-glycero-d-mannoheptose is a required intermediate for lipopolysaccharide inner-core and outer-membrane biosynthesis in several genera of pathogenic and non-pathogenic Gram-negative bacteria. ADP-l-glycero-d-mannoheptose 6-epimerase was overexpressed in E. coli and purified to apparent homogeneity by chromatographic methods. Three crystal forms of the epimerase were obtained by a hanging-drop vapor-diffusion method. A native data set for crystal form III was collected in-house on a Rigaku R-AXIS-IIC image plate at 3.0 Å resolution. The form III crystals belong to the monoclinic space group P21. The unit-cell parameters are a = 98.94, b = 110.53, c = 180.68 Å and β = 90.94°. Our recent results show that these crystals diffract to 2.0 Å resolution at the Cornell High Energy Synchrotron Source. The crystal probably contains six 40 kDa monomers per asymmetric unit, with a corresponding volume per protein mass (Vm) of 4.11 Å3 Da−1 and a solvent fraction of 70%.
ADP-l-glycero-d-mannoheptose 6-epimerase is required for lipopolysaccharide inner core biosynthesis in several genera of Gram-negative bacteria. The enzyme contains both fingerprint sequences Gly-X-Gly-X-X-Gly and Gly-X-X-Gly-X-X-Gly near its N terminus, which is indicative of an ADP binding fold. Previous studies of this ADP-l-glycero-d-mannoheptose 6-epimerase (ADP-hep 6-epimerase) were consistent with an NAD+cofactor. However, the crystal structure of this ADP-hep 6-epimerase showed bound NADP (Deacon, A. M., Ni, Y. S., Coleman, W. G., Jr., and Ealick, S. E. (2000)Structure 5, 453–462). In present studies, apo-ADP-hep 6-epimerase was reconstituted with NAD+, NADP+, and FAD. In this report we provide data that shows NAD+ and NADP+ both restored enzymatic activity, but FAD could not. Furthermore, ADP-hep 6-epimerase exhibited a preference for binding of NADP+ over NAD+. The K dvalue for NADP+ was 26 µm whereas that for NAD+ was 45 µm. Ultraviolet circular dichroism spectra showed that apo-ADP-hep 6-epimerase reconstituted with NADP+ had more secondary structure than apo-ADP-hep 6-epimerase reconstituted with NAD+. Perchloric acid extracts of the purified enzyme were assayed with NAD+-specific alcohol dehydrogenase and NADP+-specific isocitric dehydrogenase. A sample of the same perchloric acid extract was analyzed in chromatographic studies, which demonstrated that ADP-hep 6-epimerase binds NADP+ in vivo. A structural comparison of ADP-hep 6-epimerase with UDP-galactose 4-epimerase, which utilizes an NAD+cofactor, has identified the regions of ADP-hep 6-epimerase, which defines its specificity for NADP+. ADP-l-glycero-d-mannoheptose 6-epimerase is required for lipopolysaccharide inner core biosynthesis in several genera of Gram-negative bacteria. The enzyme contains both fingerprint sequences Gly-X-Gly-X-X-Gly and Gly-X-X-Gly-X-X-Gly near its N terminus, which is indicative of an ADP binding fold. Previous studies of this ADP-l-glycero-d-mannoheptose 6-epimerase (ADP-hep 6-epimerase) were consistent with an NAD+cofactor. However, the crystal structure of this ADP-hep 6-epimerase showed bound NADP (Deacon, A. M., Ni, Y. S., Coleman, W. G., Jr., and Ealick, S. E. (2000)Structure 5, 453–462). In present studies, apo-ADP-hep 6-epimerase was reconstituted with NAD+, NADP+, and FAD. In this report we provide data that shows NAD+ and NADP+ both restored enzymatic activity, but FAD could not. Furthermore, ADP-hep 6-epimerase exhibited a preference for binding of NADP+ over NAD+. The K dvalue for NADP+ was 26 µm whereas that for NAD+ was 45 µm. Ultraviolet circular dichroism spectra showed that apo-ADP-hep 6-epimerase reconstituted with NADP+ had more secondary structure than apo-ADP-hep 6-epimerase reconstituted with NAD+. Perchloric acid extracts of the purified enzyme were assayed with NAD+-specific alcohol dehydrogenase and NADP+-specific isocitric dehydrogenase. A sample of the same perchloric acid extract was analyzed in chromatographic studies, which demonstrated that ADP-hep 6-epimerase binds NADP+ in vivo. A structural comparison of ADP-hep 6-epimerase with UDP-galactose 4-epimerase, which utilizes an NAD+cofactor, has identified the regions of ADP-hep 6-epimerase, which defines its specificity for NADP+. ADP-l-glycero-d-mannoheptose 6-epimerase UDP-galactose 4-epimerase high pressure liquid chromatography ADP-l-glycero-d-mannoheptose 6-epimerase catalyzes the interconversion of ADP-d-glycero-d-mannoheptose and ADP-l-glycero-d-mannoheptose (Fig.1). This epimerization reaction is the last of four enzymatic steps required for the biosynthesis of ADP-l-glycero-d-mannoheptose. ADP-l-glycero-d-mannoheptose is the precursor of the aldoheptose, l-glycero-d-mannoheptose (or heptose), which is a highly conserved component of the lipopolysaccharide core domain among several genera of enteric and nonenteric Gram-negative bacteria (1Coleman Jr., W.G. J. Biol. Chem. 1983; 258: 1985-1990Abstract Full Text PDF PubMed Google Scholar, 2Coleman Jr., W.G. Chen L. Ding L. Galli E. Silver S. Witholt B. in Pseudomonas: Molecular Biology and Biotechnology. American Society for Microbiology, Washington, D. C.1992: 161-169Google Scholar, 3Adams G.A. Quading C. Perry M.B. Can. J. Microbiol. 1967; 13: 1605-1613Crossref PubMed Scopus (23) Google Scholar). The epimerization at carbon 6 of the heptose involves an oxidation-reduction process. In previous studies of the Escherichia coli K-12 ADP-hep 6-epimerase1 it was observed that the N terminus contained the fingerprint sequence Gly-X-Gly-X-X-Gly or Gly-X-X-Gly-X-X-Gly, which is characteristic of the ADP binding βαβαβ fold (or Rossmann fold) associated with NAD(P) binding and also FAD-binding proteins (4Pegues J.C. Chen L. Gordon A.W. Ding L. Coleman Jr., W.G. J. Bacteriol. 1990; 172: 4652-4660Crossref PubMed Google Scholar,5Bellamacina C.R. FASEB J. 1996; 10: 1257-1269Crossref PubMed Scopus (216) Google Scholar). In addition, it was observed that one nicotinamide coenzyme was tightly bound to each ADP-hep 6-epimerase subunit; enzymatic analyses suggested that NAD+ was the cofactor used by ADP-hep 6-epimerase (6Ding L. Seto B.L. Ahmed S.A. Coleman Jr., W.G. J. Biol. Chem. 1994; 269: 24384-24390Abstract Full Text PDF PubMed Google Scholar). ADP-l-glycero-d-mannoheptose 6-epimerase showed 24% sequence identity with UDP-galactose 4-epimerase (UGE) based on calculation with BLAST (7Altschul S.F. Madden T.L. Schäffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (59166) Google Scholar). The crystal structure of UGE showed two NAD molecules bound to the dimeric enzyme (8Bauer A.J. Rayment I. Frey P.A. Holden H.M. Proteins Struct. Funct. Genet. 1992; 9: 135-142Crossref Scopus (23) Google Scholar, 9Thoden J.B. Frey P.A. Holden H.M. Protein Sci. 1996; 5: 2149-2161Crossref PubMed Scopus (82) Google Scholar). However, the crystal structure of ADP-hep 6-epimerase (10Deacon A.M. Ni Y.S. Coleman Jr., W.G. Ealick S.E. Structure. 2000; 5: 453-462Abstract Full Text Full Text PDF Scopus (69) Google Scholar) indicated that NADP+ is the more likely natural cofactor for this enzyme, because only NADP was found on each of the five subunits of ADP-hep 6-epimerase. The conformation of NADP bound to ADP-hep 6-epimerase is less extended than that of NAD bound to UGE as judged by the distance between adenine C6 and C2 of the nicotinamide ring.Here we report that the enzyme ADP-l-glycero-d-mannoheptose 6-epimerase is an NADP+-dependent enzyme. NAD+ can substitute for NADP+, but enzymatic activity is reduced. Several important structural differences between ADP-hep 6-epimerase and UGE give rise to the different cofactor specificities.DISCUSSIONMany dinucleotide-binding proteins specifically require either NAD+ or NADP+ as cofactor, although some of them show a dual specificity (5Bellamacina C.R. FASEB J. 1996; 10: 1257-1269Crossref PubMed Scopus (216) Google Scholar, 16Lilley K.S. Baker P.J. Britton K.L. Stillman T.J. Brown P.E. Moir A.J.G. Engel P.C. Rice D.W. Bell J.E. Bell E. Biochim. Biophys. Acta. 1991; 1080: 191-197Crossref PubMed Scopus (34) Google Scholar, 17McPherson M.J. Wootton J.C. Nucleic Acid Res. 1983; 11: 5257-5266Crossref PubMed Scopus (71) Google Scholar, 18Moye W.S. Amuro N. Rao J.K.M. Zalkin H. J. Biol. Chem. 1985; 260: 8502-8508Abstract Full Text PDF PubMed Google Scholar, 19Scrutton N.S. Berry A. Perham R.N. Nature. 1990; 343: 38-43Crossref PubMed Scopus (644) Google Scholar, 20Wierenga R.K. Terpstra P. Hol W.G.J. J. Mol. Biol. 1986; 187: 101-107Crossref PubMed Scopus (993) Google Scholar). Our study provides strong evidence that NADP+ is the natural coenzyme of ADP-hep 6-epimerase, although NAD+ can substitute for NADP+ and still allow the epimerization reaction to proceed at a slower rate. The apparent dissociation constant determined for NADP+ versus that of NAD+ is consistent with preferential binding of NADP to ADP-hep 6-epimerase.Since the 1970s, there has been substantial interest in elucidating the fundamental basis for NAD+/NADP+ specificity of many enzymes. The fingerprint sequence Gly-X-Gly-X-X-Gly (20Wierenga R.K. Terpstra P. Hol W.G.J. J. Mol. Biol. 1986; 187: 101-107Crossref PubMed Scopus (993) Google Scholar) has been recognized as the stereotypic hallmark of dinucleotide binding, and therefore it is not surprising that the study of NAD+/NADP+ specificity has focused predominately on this sequence.To investigate the coenzyme preference of ADP-hep 6-epimerase, we have linked the biochemical and molecular modeling studies with information derived from our recent three-dimensional structural analysis. This combined approach has allowed us to (1Coleman Jr., W.G. J. Biol. Chem. 1983; 258: 1985-1990Abstract Full Text PDF PubMed Google Scholar) determine kinetic parameters that indicated a preference for NADP by ADP-hep 6-epimerase and (2Coleman Jr., W.G. Chen L. Ding L. Galli E. Silver S. Witholt B. in Pseudomonas: Molecular Biology and Biotechnology. American Society for Microbiology, Washington, D. C.1992: 161-169Google Scholar), the detailed analysis of the amino acids and structural attributes of enzyme that confer preference for nicotinamide adenine dinucleotide with or without an additional 2′-phosphate. The structural studies of ADP-l-glycero-d-mannoheptose 6-ADP-hep 6-epimerase indicate that positively charged Lys38 and Lys53 are the major contributors to the electrostatic compensation for the 2′-phosphate group of NADP. The occurrence of these positively charged residues conferred the preference of the enzyme for NADP+. This is consistent with the results of Scrutton et al. (19Scrutton N.S. Berry A. Perham R.N. Nature. 1990; 343: 38-43Crossref PubMed Scopus (644) Google Scholar), which demonstrated that the introduction of positively residues (Arg198 and Arg204) in the NAD+-binding site of E. coli glutathione reductase conferred NADP+ binding preference to the redesigned enzyme. Rizzi et al. (21Rizzi, M., Tonetti, M., Vigevani, P., Sturla, L., Bisso, A., De Flora, A., Bordo, D., and Bolognesi, M. (1998) Structure1453–1465.Google Scholar) reported in a recent structural study of GDP-4-keto-6-deoxyd-mannose epimerase/reductase that in its NADP+-binding site two positively charged residues (Arg12 and Arg36) play important roles in providing electrostatic compensation for the NADP+ ribose 2′-phosphate group.Further, residues (Gly9 and Ile11) present in the Gly-X-X-Gly-X-X-Gly motif (21Rizzi, M., Tonetti, M., Vigevani, P., Sturla, L., Bisso, A., De Flora, A., Bordo, D., and Bolognesi, M. (1998) Structure1453–1465.Google Scholar) are positioned near the diphosphate bridge of the ADP-hep 6-epimerase-bound NADP. Residues Ser116, Tyr140, and Lys144, thought to be involved in the catalytic mechanism of ADP-hep 6-epimerase, are located near the nicotinamide ring and its attached ribose. Thus, there exists a significant degree of structural and sequence similarity for the NAD(P)+-binding sites of ADP-hep 6-epimerase and other NAD(P)-binding proteins. Further mutagenesis studies will be used to provide additional insight into the cofactor specificity of ADP-hep 6-epimerase and to determine in more detail the significance of the residues involved. ADP-l-glycero-d-mannoheptose 6-epimerase catalyzes the interconversion of ADP-d-glycero-d-mannoheptose and ADP-l-glycero-d-mannoheptose (Fig.1). This epimerization reaction is the last of four enzymatic steps required for the biosynthesis of ADP-l-glycero-d-mannoheptose. ADP-l-glycero-d-mannoheptose is the precursor of the aldoheptose, l-glycero-d-mannoheptose (or heptose), which is a highly conserved component of the lipopolysaccharide core domain among several genera of enteric and nonenteric Gram-negative bacteria (1Coleman Jr., W.G. J. Biol. Chem. 1983; 258: 1985-1990Abstract Full Text PDF PubMed Google Scholar, 2Coleman Jr., W.G. Chen L. Ding L. Galli E. Silver S. Witholt B. in Pseudomonas: Molecular Biology and Biotechnology. American Society for Microbiology, Washington, D. C.1992: 161-169Google Scholar, 3Adams G.A. Quading C. Perry M.B. Can. J. Microbiol. 1967; 13: 1605-1613Crossref PubMed Scopus (23) Google Scholar). The epimerization at carbon 6 of the heptose involves an oxidation-reduction process. In previous studies of the Escherichia coli K-12 ADP-hep 6-epimerase1 it was observed that the N terminus contained the fingerprint sequence Gly-X-Gly-X-X-Gly or Gly-X-X-Gly-X-X-Gly, which is characteristic of the ADP binding βαβαβ fold (or Rossmann fold) associated with NAD(P) binding and also FAD-binding proteins (4Pegues J.C. Chen L. Gordon A.W. Ding L. Coleman Jr., W.G. J. Bacteriol. 1990; 172: 4652-4660Crossref PubMed Google Scholar,5Bellamacina C.R. FASEB J. 1996; 10: 1257-1269Crossref PubMed Scopus (216) Google Scholar). In addition, it was observed that one nicotinamide coenzyme was tightly bound to each ADP-hep 6-epimerase subunit; enzymatic analyses suggested that NAD+ was the cofactor used by ADP-hep 6-epimerase (6Ding L. Seto B.L. Ahmed S.A. Coleman Jr., W.G. J. Biol. Chem. 1994; 269: 24384-24390Abstract Full Text PDF PubMed Google Scholar). ADP-l-glycero-d-mannoheptose 6-epimerase showed 24% sequence identity with UDP-galactose 4-epimerase (UGE) based on calculation with BLAST (7Altschul S.F. Madden T.L. Schäffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (59166) Google Scholar). The crystal structure of UGE showed two NAD molecules bound to the dimeric enzyme (8Bauer A.J. Rayment I. Frey P.A. Holden H.M. Proteins Struct. Funct. Genet. 1992; 9: 135-142Crossref Scopus (23) Google Scholar, 9Thoden J.B. Frey P.A. Holden H.M. Protein Sci. 1996; 5: 2149-2161Crossref PubMed Scopus (82) Google Scholar). However, the crystal structure of ADP-hep 6-epimerase (10Deacon A.M. Ni Y.S. Coleman Jr., W.G. Ealick S.E. Structure. 2000; 5: 453-462Abstract Full Text Full Text PDF Scopus (69) Google Scholar) indicated that NADP+ is the more likely natural cofactor for this enzyme, because only NADP was found on each of the five subunits of ADP-hep 6-epimerase. The conformation of NADP bound to ADP-hep 6-epimerase is less extended than that of NAD bound to UGE as judged by the distance between adenine C6 and C2 of the nicotinamide ring. Here we report that the enzyme ADP-l-glycero-d-mannoheptose 6-epimerase is an NADP+-dependent enzyme. NAD+ can substitute for NADP+, but enzymatic activity is reduced. Several important structural differences between ADP-hep 6-epimerase and UGE give rise to the different cofactor specificities. DISCUSSIONMany dinucleotide-binding proteins specifically require either NAD+ or NADP+ as cofactor, although some of them show a dual specificity (5Bellamacina C.R. FASEB J. 1996; 10: 1257-1269Crossref PubMed Scopus (216) Google Scholar, 16Lilley K.S. Baker P.J. Britton K.L. Stillman T.J. Brown P.E. Moir A.J.G. Engel P.C. Rice D.W. Bell J.E. Bell E. Biochim. Biophys. Acta. 1991; 1080: 191-197Crossref PubMed Scopus (34) Google Scholar, 17McPherson M.J. Wootton J.C. Nucleic Acid Res. 1983; 11: 5257-5266Crossref PubMed Scopus (71) Google Scholar, 18Moye W.S. Amuro N. Rao J.K.M. Zalkin H. J. Biol. Chem. 1985; 260: 8502-8508Abstract Full Text PDF PubMed Google Scholar, 19Scrutton N.S. Berry A. Perham R.N. Nature. 1990; 343: 38-43Crossref PubMed Scopus (644) Google Scholar, 20Wierenga R.K. Terpstra P. Hol W.G.J. J. Mol. Biol. 1986; 187: 101-107Crossref PubMed Scopus (993) Google Scholar). Our study provides strong evidence that NADP+ is the natural coenzyme of ADP-hep 6-epimerase, although NAD+ can substitute for NADP+ and still allow the epimerization reaction to proceed at a slower rate. The apparent dissociation constant determined for NADP+ versus that of NAD+ is consistent with preferential binding of NADP to ADP-hep 6-epimerase.Since the 1970s, there has been substantial interest in elucidating the fundamental basis for NAD+/NADP+ specificity of many enzymes. The fingerprint sequence Gly-X-Gly-X-X-Gly (20Wierenga R.K. Terpstra P. Hol W.G.J. J. Mol. Biol. 1986; 187: 101-107Crossref PubMed Scopus (993) Google Scholar) has been recognized as the stereotypic hallmark of dinucleotide binding, and therefore it is not surprising that the study of NAD+/NADP+ specificity has focused predominately on this sequence.To investigate the coenzyme preference of ADP-hep 6-epimerase, we have linked the biochemical and molecular modeling studies with information derived from our recent three-dimensional structural analysis. This combined approach has allowed us to (1Coleman Jr., W.G. J. Biol. Chem. 1983; 258: 1985-1990Abstract Full Text PDF PubMed Google Scholar) determine kinetic parameters that indicated a preference for NADP by ADP-hep 6-epimerase and (2Coleman Jr., W.G. Chen L. Ding L. Galli E. Silver S. Witholt B. in Pseudomonas: Molecular Biology and Biotechnology. American Society for Microbiology, Washington, D. C.1992: 161-169Google Scholar), the detailed analysis of the amino acids and structural attributes of enzyme that confer preference for nicotinamide adenine dinucleotide with or without an additional 2′-phosphate. The structural studies of ADP-l-glycero-d-mannoheptose 6-ADP-hep 6-epimerase indicate that positively charged Lys38 and Lys53 are the major contributors to the electrostatic compensation for the 2′-phosphate group of NADP. The occurrence of these positively charged residues conferred the preference of the enzyme for NADP+. This is consistent with the results of Scrutton et al. (19Scrutton N.S. Berry A. Perham R.N. Nature. 1990; 343: 38-43Crossref PubMed Scopus (644) Google Scholar), which demonstrated that the introduction of positively residues (Arg198 and Arg204) in the NAD+-binding site of E. coli glutathione reductase conferred NADP+ binding preference to the redesigned enzyme. Rizzi et al. (21Rizzi, M., Tonetti, M., Vigevani, P., Sturla, L., Bisso, A., De Flora, A., Bordo, D., and Bolognesi, M. (1998) Structure1453–1465.Google Scholar) reported in a recent structural study of GDP-4-keto-6-deoxyd-mannose epimerase/reductase that in its NADP+-binding site two positively charged residues (Arg12 and Arg36) play important roles in providing electrostatic compensation for the NADP+ ribose 2′-phosphate group.Further, residues (Gly9 and Ile11) present in the Gly-X-X-Gly-X-X-Gly motif (21Rizzi, M., Tonetti, M., Vigevani, P., Sturla, L., Bisso, A., De Flora, A., Bordo, D., and Bolognesi, M. (1998) Structure1453–1465.Google Scholar) are positioned near the diphosphate bridge of the ADP-hep 6-epimerase-bound NADP. Residues Ser116, Tyr140, and Lys144, thought to be involved in the catalytic mechanism of ADP-hep 6-epimerase, are located near the nicotinamide ring and its attached ribose. Thus, there exists a significant degree of structural and sequence similarity for the NAD(P)+-binding sites of ADP-hep 6-epimerase and other NAD(P)-binding proteins. Further mutagenesis studies will be used to provide additional insight into the cofactor specificity of ADP-hep 6-epimerase and to determine in more detail the significance of the residues involved. Many dinucleotide-binding proteins specifically require either NAD+ or NADP+ as cofactor, although some of them show a dual specificity (5Bellamacina C.R. FASEB J. 1996; 10: 1257-1269Crossref PubMed Scopus (216) Google Scholar, 16Lilley K.S. Baker P.J. Britton K.L. Stillman T.J. Brown P.E. Moir A.J.G. Engel P.C. Rice D.W. Bell J.E. Bell E. Biochim. Biophys. Acta. 1991; 1080: 191-197Crossref PubMed Scopus (34) Google Scholar, 17McPherson M.J. Wootton J.C. Nucleic Acid Res. 1983; 11: 5257-5266Crossref PubMed Scopus (71) Google Scholar, 18Moye W.S. Amuro N. Rao J.K.M. Zalkin H. J. Biol. Chem. 1985; 260: 8502-8508Abstract Full Text PDF PubMed Google Scholar, 19Scrutton N.S. Berry A. Perham R.N. Nature. 1990; 343: 38-43Crossref PubMed Scopus (644) Google Scholar, 20Wierenga R.K. Terpstra P. Hol W.G.J. J. Mol. Biol. 1986; 187: 101-107Crossref PubMed Scopus (993) Google Scholar). Our study provides strong evidence that NADP+ is the natural coenzyme of ADP-hep 6-epimerase, although NAD+ can substitute for NADP+ and still allow the epimerization reaction to proceed at a slower rate. The apparent dissociation constant determined for NADP+ versus that of NAD+ is consistent with preferential binding of NADP to ADP-hep 6-epimerase. Since the 1970s, there has been substantial interest in elucidating the fundamental basis for NAD+/NADP+ specificity of many enzymes. The fingerprint sequence Gly-X-Gly-X-X-Gly (20Wierenga R.K. Terpstra P. Hol W.G.J. J. Mol. Biol. 1986; 187: 101-107Crossref PubMed Scopus (993) Google Scholar) has been recognized as the stereotypic hallmark of dinucleotide binding, and therefore it is not surprising that the study of NAD+/NADP+ specificity has focused predominately on this sequence. To investigate the coenzyme preference of ADP-hep 6-epimerase, we have linked the biochemical and molecular modeling studies with information derived from our recent three-dimensional structural analysis. This combined approach has allowed us to (1Coleman Jr., W.G. J. Biol. Chem. 1983; 258: 1985-1990Abstract Full Text PDF PubMed Google Scholar) determine kinetic parameters that indicated a preference for NADP by ADP-hep 6-epimerase and (2Coleman Jr., W.G. Chen L. Ding L. Galli E. Silver S. Witholt B. in Pseudomonas: Molecular Biology and Biotechnology. American Society for Microbiology, Washington, D. C.1992: 161-169Google Scholar), the detailed analysis of the amino acids and structural attributes of enzyme that confer preference for nicotinamide adenine dinucleotide with or without an additional 2′-phosphate. The structural studies of ADP-l-glycero-d-mannoheptose 6-ADP-hep 6-epimerase indicate that positively charged Lys38 and Lys53 are the major contributors to the electrostatic compensation for the 2′-phosphate group of NADP. The occurrence of these positively charged residues conferred the preference of the enzyme for NADP+. This is consistent with the results of Scrutton et al. (19Scrutton N.S. Berry A. Perham R.N. Nature. 1990; 343: 38-43Crossref PubMed Scopus (644) Google Scholar), which demonstrated that the introduction of positively residues (Arg198 and Arg204) in the NAD+-binding site of E. coli glutathione reductase conferred NADP+ binding preference to the redesigned enzyme. Rizzi et al. (21Rizzi, M., Tonetti, M., Vigevani, P., Sturla, L., Bisso, A., De Flora, A., Bordo, D., and Bolognesi, M. (1998) Structure1453–1465.Google Scholar) reported in a recent structural study of GDP-4-keto-6-deoxyd-mannose epimerase/reductase that in its NADP+-binding site two positively charged residues (Arg12 and Arg36) play important roles in providing electrostatic compensation for the NADP+ ribose 2′-phosphate group. Further, residues (Gly9 and Ile11) present in the Gly-X-X-Gly-X-X-Gly motif (21Rizzi, M., Tonetti, M., Vigevani, P., Sturla, L., Bisso, A., De Flora, A., Bordo, D., and Bolognesi, M. (1998) Structure1453–1465.Google Scholar) are positioned near the diphosphate bridge of the ADP-hep 6-epimerase-bound NADP. Residues Ser116, Tyr140, and Lys144, thought to be involved in the catalytic mechanism of ADP-hep 6-epimerase, are located near the nicotinamide ring and its attached ribose. Thus, there exists a significant degree of structural and sequence similarity for the NAD(P)+-binding sites of ADP-hep 6-epimerase and other NAD(P)-binding proteins. Further mutagenesis studies will be used to provide additional insight into the cofactor specificity of ADP-hep 6-epimerase and to determine in more detail the significance of the residues involved.
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