cDNA from rat cells with reconstitutive galactose-epimerase activity in<i>E. coli</i>

Zeschnigk, Michael; Wilcken‐Bergmann, Brigitte von; Starzinski‐Powitz, Anna

doi:10.1093/nar/18.17.5289

Cited by 9 publications

(5 citation statements)

References 3 publications

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“…d Saccharomyces cereVisiae UDPgalactose 4-epimerase (Citron & Donelson, 1984). e Rat myoblast UDPgalactose 4-epimerase (Zeschnigk et al, 1990). f Streptomyces liVidans UDP-galactose 4-epimerase (Adams et al, 1988).…”

Section: Discussionmentioning

confidence: 99%

Probing the Coenzyme and Substrate Binding Events of CDP-d-glucose 4,6-Dehydratase: Mechanistic Implications

Thorson

Liu

1996

Biochemistry

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NAD+-dependent nucleotidyl diphosphohexose 4,6-dehydratases which transform nucleotidyl diphosphohexoses into corresponding 4-keto-6-deoxy sugar derivatives are essential to the formation of all 6-deoxyhexoses. Studies of the CDP-D-glucose 4,6-dehydratase (Eod) from Yersinia had shown that this dimeric protein binds only 1 equiv of NAD+/mol of enzyme and, unlike other enzymes of the same class, displays a unique NAD+ requirement for full catalytic activity. Analysis of the primary sequence revealed an extended ADP-binding fold (GHTGFKG) which deviates from the common Rossman consensus (GXGXXG) and thus may have contributed to Eod's limited NAD+ affinity. In particular, the presence of His17 in the beta-turn region and that of Lys21 in a position typically occupied by a small hydrophobic residue may impose electronic or steric perturbations to this essential binding motif. To better understand the correlation between the binding properties and primary sequence, mutants (H17G and K21I) were constructed to provide enzymes containing an ADP binding region which more closely resembles the Rossman-type fold. Analysis of the cofactor and substrate binding characteristics of the wild-type and mutant enzymes helped define the presence of two binding sites for both CDP-d_glucose and NAD+ per enzyme molecule. While both mutants displayed enhanced NAD+ affinity, the H17G mutation resulted in an enzyme with slightly higher kcat and a 3-fold increase in catalytic efficiency (kcat/Km). The large anticooperativity found for NAD+ binding (K1=40.3 + or - 0.4 nM, K2=539.8 + or - 4.8 nM) may explain why the cofactor binding sites of wild-type Eod are only half-occupied. Further examination also revealed the purified Eod to contain sequestered NADH and that the affinity of Eod for NADH(K1=0.21 + or - 0.01 nM, K2= 7.46 + or -0.25 nM) is much higher than that for NAD+. Thus, it is possible that Eod's half-site saturation of NAD+ per enzyme dimer may also be attributed to a significant portion of the cofactor binding sites being occupied by NADH. Interestingly, the sequestered NADH is released upon binding with CDP-D-glucose. These results implicate a new kinetic mechanism for Eod catalysis.

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Section: Discussionmentioning

confidence: 99%

Probing the Coenzyme and Substrate Binding Events of CDP-d-glucose 4,6-Dehydratase: Mechanistic Implications

Thorson

Liu

1996

Biochemistry

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“…An identity of 66.7% was observed between amino acids 7 to 18 of the VipA sequence and the corresponding GDP-mannose dehydrogenase sequence. The VipB protein showed limited similarity to UDP-glucose 4-epimerase from prokaryotic (1, 31, 38) and eukaryotic (7,50) origins. There was 28.9% identity of VipB and UDP-glucose 4-epimerase from E. coli (31,33) over 211 amino acids (Fig.…”

Section: Methodsmentioning

confidence: 99%

Complete nucleotide sequence and molecular characterization of ViaB region encoding Vi antigen in Salmonella typhi

et al. 1993

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Plasmid pGBM124, which contains a 14-kb Sablonella typhi chromosomal DNA fragment capable of producing the Vi antigen in Escherichia coli HB101 and ViaB-deleted S. typhi GIFU 10007-3, was studied. We determined the complete nucleotide sequence of this fragment and found 11 open reading frames. Mutagenesis, subcloning, and complementation analysis showed that three genes (vipA, vipB, and vipC) are involved in biosynthesis of the Vi polysaccharide. The putative primary amino acid sequence suggests that both vipA and vipB encode the NAD-or NADP-dependent enzymes to synthesize the nucleotide sugar for the Vi polysaccharide. Five genes (vexA, vexB, vexC, vexD, and vexE) may be involved in translocation of the Vi polysaccharide.Proteins VexA, VexB, VexC, and VexD had moderate similarities to components of group II capsule transporters, and the VexC protein had a putative ATP-binding site. These data indicate that the transport system for the Vi polysaccharide belongs to the ATP-binding cassette transporters. By using the isogenic Vi+ and Vi-strains constructed in this study, we reconfirmed that the Vi antigen is necessary for the serum resistance of S. lyphi.

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“…As a first test of the ability of plasmid-encoded mammalian GALE enzymes to function in yeast, yBBQ1 cells were transformed with yeast low-copy number plasmids encoding either wild-type rat (Zeschnigk et al 1990) or hGALE. Both wild-type yeast GALl 0 and plasmid backbone alone also were introduced as controls.…”

Section: A Yeast Expression System For Hgalementioning

confidence: 99%

Characterization of Two Mutations Associated with Epimerase-Deficiency Galactosemia, by Use of a Yeast Expression System for Human UDP-Galactose-4-Epimerase

Quimby¹,

Alano

Almashanu

et al. 1997

The American Journal of Human Genetics

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UDP-galactose-4-epimerase (GALE) is a highly conserved enzyme that catalyzes the interconversion of UDP-galactose and UDP-glucose. Impairment of this enzyme in humans results in one of two clinically distinct forms of epimerase-deficiency galactosemia-one benign, the other severe. The molecular and biochemical distinction between these disorders remains unknown. To enable structural and functional studies of both wild-type and patient-derived alleles of human GALE (hGALE), we have developed and applied a null-background yeast expression system for the human enzyme. We have demonstrated that wild-type hGALE sequences phenotypically complement a yeast gal10 deletion, and we have biochemically characterized the wild-type human enzyme isolated from these cells. Furthermore, we have expressed and characterized two mutant alleles, L183P-hGALE and N34S-hGALE, both derived from a patient with no detectable GALE activity in red blood cells but with approximately 14% activity in cultured lymphoblasts. Analyses of crude extracts of yeast expressing L183P-hGALE demonstrated 4% wild-type activity and 6% wild-type abundance. Extracts of yeast expressing N34S-hGALE demonstrated approximately 70% wild-type activity and normal abundance. However, yeast coexpressing both L183P-hGALE and N34S-hGALE exhibited only approximately 7% wild-type levels of activity, thereby confirming the functional impact of both substitutions and raising the intriguing possibility that some form of dominant-negative interaction may exist between the mutant alleles found in this patient. The results reported here establish the utility of the yeast-based hGALE-expression system and set the stage for more-detailed studies of this important enzyme and its role in epimerase-deficiency galactosemia.

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cDNA from rat cells with reconstitutive galactose-epimerase activity inE. coli

Cited by 9 publications

References 3 publications

Probing the Coenzyme and Substrate Binding Events of CDP-d-glucose 4,6-Dehydratase: Mechanistic Implications

Probing the Coenzyme and Substrate Binding Events of CDP-d-glucose 4,6-Dehydratase: Mechanistic Implications

Complete nucleotide sequence and molecular characterization of ViaB region encoding Vi antigen in Salmonella typhi

Characterization of Two Mutations Associated with Epimerase-Deficiency Galactosemia, by Use of a Yeast Expression System for Human UDP-Galactose-4-Epimerase

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