FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1974616082> ?p ?o ?g. }

• W1974616082 endingPage "2626" @default.
• W1974616082 startingPage "2619" @default.
• W1974616082 abstract "Porphobilinogen synthase (PBGS) is an ancient enzyme essential to tetrapyrrole biosynthesis (e.g. heme, chlorophyll, and vitamin B12). Two common alleles encoding human PBGS, K59 and N59, have been correlated with differential susceptibility of humans to lead poisoning. However, a model for human PBGS based on homologous crystal structures shows the location of the allelic variation to be distant from the active site with its two Zn(II). Previous microbial expression systems for human PBGS have resulted in a poor yield. Here, an artificial gene encoding human PBGS was constructed by recursive polymerase chain reaction from synthetic oligonucleotides to rectify this problem. The artificial gene was made to resemble the highly expressed homologous Escherichia coli hemB gene and to remove rare codons that can confound heterologous protein expression in E. coli. We have expressed and purified recombinant human PBGS variants K59 and N59 in 100-mg quantities. Both human PBGS proteins purified with eight Zn(II)/octamer; Zn(II) binding was shown to be pH-dependent; and Pb(II) could displace some of the Zn(II). However, there was no differential displacement of Zn(II) by Pb(II) between K59 and N59, and simple Pb(II) inhibition studies revealed no allelic difference. Porphobilinogen synthase (PBGS) is an ancient enzyme essential to tetrapyrrole biosynthesis (e.g. heme, chlorophyll, and vitamin B12). Two common alleles encoding human PBGS, K59 and N59, have been correlated with differential susceptibility of humans to lead poisoning. However, a model for human PBGS based on homologous crystal structures shows the location of the allelic variation to be distant from the active site with its two Zn(II). Previous microbial expression systems for human PBGS have resulted in a poor yield. Here, an artificial gene encoding human PBGS was constructed by recursive polymerase chain reaction from synthetic oligonucleotides to rectify this problem. The artificial gene was made to resemble the highly expressed homologous Escherichia coli hemB gene and to remove rare codons that can confound heterologous protein expression in E. coli. We have expressed and purified recombinant human PBGS variants K59 and N59 in 100-mg quantities. Both human PBGS proteins purified with eight Zn(II)/octamer; Zn(II) binding was shown to be pH-dependent; and Pb(II) could displace some of the Zn(II). However, there was no differential displacement of Zn(II) by Pb(II) between K59 and N59, and simple Pb(II) inhibition studies revealed no allelic difference. porphobilinogen synthase polymerase chain reaction isopropyl-β-d-thiogalactopyranoside phenylmethylsulfonyl fluoride Human genetic polymorphisms are increasingly being correlated with disease states through epidemiological analysis. These types of studies lay the groundwork for the extended practice of preventive medicine. A common genetic polymorphism in the human ALADgene, 1The naturally occurring human gene is called ALAD based on the commonly used alternative enzyme name 5-aminolevulinic-aciddehydratase. 1The naturally occurring human gene is called ALAD based on the commonly used alternative enzyme name 5-aminolevulinic-aciddehydratase. encoding the enzyme porphobilinogen synthase (PBGS2; EC 4.2.1.24), has been correlated with susceptibility to the environmental toxin lead (1.Wetmur J.G. Lehnert G. Desnick R.J. Environ. Res. 1991; 56: 109-119Crossref PubMed Scopus (139) Google Scholar, 2.Schwartz B.S. Lee B.K. Stewart W. Ahn K.D. Springer K. Kelsey K. Am. J. Epidemiol. 1995; 142: 738-745Crossref PubMed Scopus (101) Google Scholar, 3.Smith C.M. Wang X. Hu H. Kelsey K.T. Environ. Health Perspect. 1995; 103: 248-253PubMed Google Scholar, 4.Bergdahl I.A. Gerhardsson L. Schutz A. Desnick R.J. Wetmur J.G. Skerfving S. Arch. Environ. Health. 1997; 52: 91-96Crossref PubMed Scopus (66) Google Scholar, 5.Bergdahl I.A. Grubb A. Schutz A. Desnick R.J. Wetmur J.G. Sassa S. Skerfving S. Pharmacol. Toxicol. 1997; 81: 153-158Crossref PubMed Scopus (155) Google Scholar, 6.Fleming D.E. Chettle D.R. Wetmur J.G. Desnick R.J. Robin J.P. Boulay D. Richard N.S. Gordon C.L. Webber C.E. Environ. Res. 1998; 77: 49-61Crossref PubMed Scopus (74) Google Scholar). As lead poisoning is the most common preventable childhood neurological environmental disease, correlation of the epidemiological results with protein structure/function studies is in order.PBGS catalyzes the first common step in the biosynthesis of all tetrapyrroles (heme, chlorophyll, vitamin B12, cofactor F430, etc.). Human PBGS is a Zn(II) metalloenzyme unique in its sensitivity to inhibition by lead. Although all PBGSs appear to be metalloenzymes (7.Shemin D. Boyer P. The Enzymes. 3rd Ed. VII. Academic Press, New York1972: 232-237Google Scholar), metal ion usage varies dramatically between species (8.Petrovich R.M. Litwin S. Jaffe E.K. J. Biol. Chem. 1996; 271: 8692-8699Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 9.Frankenberg N. Jahn D. Jaffe E.K. Biochemistry. 1999; 38: 13976-13982Crossref PubMed Scopus (24) Google Scholar). One outcome of this variation is that microbial and plant PBGSs are poor models for studying the effect of lead on human PBGS function. Lead inhibition of human PBGS is one of the earliest physiological responses to lead intoxication and as such is believed to be related to the detrimental effects of low level lead poisoning. However, mapping the common human polymorphism onto the x-ray crystal structure of the related yeast PBGS protein does not indicate a structural variation that would obviously affect either metal binding or catalytic function. A model for human PBGS is presented in Fig.1 and shows the location of amino acid 59, which is lysine in the ALAD1 gene product and asparagine in the ALAD2 gene product.Production of human PBGS for study has been problematic. Purification of PBGS from human blood gives relatively low yield, is plagued by considerations of blood-borne diseases, and contains a mixture of the isozymes (10.Anderson P.M. Desnick R.J. J. Biol. Chem. 1979; 254: 6924-6930Abstract Full Text PDF PubMed Google Scholar, 11.Gibbs P.N. Chaudhry A.G. Jordan P.M. Biochem. J. 1985; 230: 25-34Crossref PubMed Scopus (34) Google Scholar). The human ALAD gene was cloned and sequenced more than a decade ago and found to express poorly inEscherichia coli (12.Wetmur J.G. Bishop D.F. Cantelmo C. Desnick R.J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7703-7707Crossref PubMed Scopus (100) Google Scholar). To generate a better expression system, the gene was cloned into yeast (13.Schauer W.E. Mattoon J.R. Curr. Genet. 1990; 17: 1-6Crossref PubMed Scopus (4) Google Scholar), but the levels of protein expression remain insufficient for thorough functional analysis.For a novel approach to heterologous expression of human PBGS, we chose to mimic a construct used for overexpression of the E. coli hemB gene in E. coli (14.Roessner C.A. Spencer J.B. Ozaki S. Min C. Atshaves B.P. Nayar P. Anousis N. Stolowich N.J. Holderman M.T. Scott A.I. Protein Expression Purif. 1995; 6: 155-163Crossref PubMed Scopus (29) Google Scholar, 15.Mitchell L.W. Jaffe E.K. Arch. Biochem. Biophys. 1993; 300: 169-177Crossref PubMed Scopus (50) Google Scholar). This system can generate hundreds of milligrams of E. coli PBGS (up to 30% of the soluble protein). The reasons for high level expression are not fully understood. In this case, the hemB gene is apparently downstream from a naturally strong E. coli promoter, and other poorly understood aspects of gene structure may contribute to the phenomenal levels of constitutive expression observed. A second consideration in artificial gene design is codon usage. The humanALAD gene was analyzed and found to contain clusters of codons that are rarely used by E. coli (see Fig.2 A). In contrast, the E. coli hemB gene contains only one rare codon. Kane (16.Kane J.F. Curr. Opin. Biotechnol. 1995; 6: 494-500Crossref PubMed Scopus (594) Google Scholar) has described how clusters of six specific rare codons can be detrimental to both the quality and quantity of heterologous proteins expressed in E. coli, and specific translational errors have been documented for such clusters (17.Forman M.D. Stack R.F. Masters P.S. Hauer C.R. Baxter S.M. Protein Sci. 1998; 7: 500-503Crossref PubMed Scopus (45) Google Scholar). Hence, in the design of an artificial gene for human PBGS, theE. coli hemB gene structure was mimicked to the greatest extent possible, and rare codons were replaced.Figure 2From ALAD to the artificial gene for human PBGS. A, the cDNA sequence encoding human PBGS (GenBankTM accession number M13928) is aligned with the encoded amino acid sequence (12.Wetmur J.G. Bishop D.F. Cantelmo C. Desnick R.J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7703-7707Crossref PubMed Scopus (100) Google Scholar). Regions of amino acid identity to E. coli PBGS are shaded (131 out of 330 codons). The six most detrimental E. coli codons (16.Kane J.F. Curr. Opin. Biotechnol. 1995; 6: 494-500Crossref PubMed Scopus (594) Google Scholar) with their encoded amino acids are in large type (20 out of 330 codons). Concerns for the quantity or quality of the expressed protein stem from clusters of these rare codons. All codonsshaded and/or in large type were subject to change in the design of the artificial gene according to the rationale described under “Experimental Procedures.” B–F, shown are maps of the DNA constructs used in this study. B, plasmid pLM1228 was originally prepared for expression of site-directed mutants of E. coli PBGS. The E. coli hemB gene and its flanking DNA were cloned into the EcoRI site of pUC119 (14.Roessner C.A. Spencer J.B. Ozaki S. Min C. Atshaves B.P. Nayar P. Anousis N. Stolowich N.J. Holderman M.T. Scott A.I. Protein Expression Purif. 1995; 6: 155-163Crossref PubMed Scopus (29) Google Scholar). Section I is part of the naturalhemB promoter region; section II is the coding region of hemB; section III is 3′-untranslated DNA; and section IV is part of theyaiG gene. For unknown reasons, this construct gives high constitutive expression of E. coli PBGS in a variety ofE. coli hosts. C, the PCR target EJhumresembles its cognate portion of pLM1228 except that the artificial gene encodes human PBGS (see “Experimental Procedures”).D, the plasmid pEJhum, designed for constitutive expression of the artificial gene, did not yield stable constitutive expression.E, the PCR target MVhum contains the coding region of the artificial gene alone plus the NdeI andBamHI sites (and some flanking DNA) for insertion into pET3a or pET11a. F, the final plasmid pMVhum contains the artificial gene under control of T7 polymerase in a pET3 background.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Here we describe the design and synthesis of a PCR product (EJhum) containing an artificial gene coding for human PBGS.EJhum mimics a pUC119 version of pCR261 (14.Roessner C.A. Spencer J.B. Ozaki S. Min C. Atshaves B.P. Nayar P. Anousis N. Stolowich N.J. Holderman M.T. Scott A.I. Protein Expression Purif. 1995; 6: 155-163Crossref PubMed Scopus (29) Google Scholar), which gives high level constitutive expression of the E. coli hemB gene (encoding E. coli PBGS) in a variety of E. colihost strains. Successful large-scale protein production of human PBGS was obtained by expression under control of the T7 polymerase system using a plasmid named pMVhum, which encodes the K59 protein corresponding to the ALAD1 allele inserted into the pET3 vector. We present the expression and purification of human PBGS from this construct and a basic characterization of the two human PBGS proteins K59 and N59, the latter a product of site-directed mutagenesis. We also compare the Zn(II) binding properties of the two proteins and address the ability of Pb(II) to displace the essential Zn(II). Human genetic polymorphisms are increasingly being correlated with disease states through epidemiological analysis. These types of studies lay the groundwork for the extended practice of preventive medicine. A common genetic polymorphism in the human ALADgene, 1The naturally occurring human gene is called ALAD based on the commonly used alternative enzyme name 5-aminolevulinic-aciddehydratase. 1The naturally occurring human gene is called ALAD based on the commonly used alternative enzyme name 5-aminolevulinic-aciddehydratase. encoding the enzyme porphobilinogen synthase (PBGS2; EC 4.2.1.24), has been correlated with susceptibility to the environmental toxin lead (1.Wetmur J.G. Lehnert G. Desnick R.J. Environ. Res. 1991; 56: 109-119Crossref PubMed Scopus (139) Google Scholar, 2.Schwartz B.S. Lee B.K. Stewart W. Ahn K.D. Springer K. Kelsey K. Am. J. Epidemiol. 1995; 142: 738-745Crossref PubMed Scopus (101) Google Scholar, 3.Smith C.M. Wang X. Hu H. Kelsey K.T. Environ. Health Perspect. 1995; 103: 248-253PubMed Google Scholar, 4.Bergdahl I.A. Gerhardsson L. Schutz A. Desnick R.J. Wetmur J.G. Skerfving S. Arch. Environ. Health. 1997; 52: 91-96Crossref PubMed Scopus (66) Google Scholar, 5.Bergdahl I.A. Grubb A. Schutz A. Desnick R.J. Wetmur J.G. Sassa S. Skerfving S. Pharmacol. Toxicol. 1997; 81: 153-158Crossref PubMed Scopus (155) Google Scholar, 6.Fleming D.E. Chettle D.R. Wetmur J.G. Desnick R.J. Robin J.P. Boulay D. Richard N.S. Gordon C.L. Webber C.E. Environ. Res. 1998; 77: 49-61Crossref PubMed Scopus (74) Google Scholar). As lead poisoning is the most common preventable childhood neurological environmental disease, correlation of the epidemiological results with protein structure/function studies is in order. PBGS catalyzes the first common step in the biosynthesis of all tetrapyrroles (heme, chlorophyll, vitamin B12, cofactor F430, etc.). Human PBGS is a Zn(II) metalloenzyme unique in its sensitivity to inhibition by lead. Although all PBGSs appear to be metalloenzymes (7.Shemin D. Boyer P. The Enzymes. 3rd Ed. VII. Academic Press, New York1972: 232-237Google Scholar), metal ion usage varies dramatically between species (8.Petrovich R.M. Litwin S. Jaffe E.K. J. Biol. Chem. 1996; 271: 8692-8699Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 9.Frankenberg N. Jahn D. Jaffe E.K. Biochemistry. 1999; 38: 13976-13982Crossref PubMed Scopus (24) Google Scholar). One outcome of this variation is that microbial and plant PBGSs are poor models for studying the effect of lead on human PBGS function. Lead inhibition of human PBGS is one of the earliest physiological responses to lead intoxication and as such is believed to be related to the detrimental effects of low level lead poisoning. However, mapping the common human polymorphism onto the x-ray crystal structure of the related yeast PBGS protein does not indicate a structural variation that would obviously affect either metal binding or catalytic function. A model for human PBGS is presented in Fig.1 and shows the location of amino acid 59, which is lysine in the ALAD1 gene product and asparagine in the ALAD2 gene product. Production of human PBGS for study has been problematic. Purification of PBGS from human blood gives relatively low yield, is plagued by considerations of blood-borne diseases, and contains a mixture of the isozymes (10.Anderson P.M. Desnick R.J. J. Biol. Chem. 1979; 254: 6924-6930Abstract Full Text PDF PubMed Google Scholar, 11.Gibbs P.N. Chaudhry A.G. Jordan P.M. Biochem. J. 1985; 230: 25-34Crossref PubMed Scopus (34) Google Scholar). The human ALAD gene was cloned and sequenced more than a decade ago and found to express poorly inEscherichia coli (12.Wetmur J.G. Bishop D.F. Cantelmo C. Desnick R.J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7703-7707Crossref PubMed Scopus (100) Google Scholar). To generate a better expression system, the gene was cloned into yeast (13.Schauer W.E. Mattoon J.R. Curr. Genet. 1990; 17: 1-6Crossref PubMed Scopus (4) Google Scholar), but the levels of protein expression remain insufficient for thorough functional analysis. For a novel approach to heterologous expression of human PBGS, we chose to mimic a construct used for overexpression of the E. coli hemB gene in E. coli (14.Roessner C.A. Spencer J.B. Ozaki S. Min C. Atshaves B.P. Nayar P. Anousis N. Stolowich N.J. Holderman M.T. Scott A.I. Protein Expression Purif. 1995; 6: 155-163Crossref PubMed Scopus (29) Google Scholar, 15.Mitchell L.W. Jaffe E.K. Arch. Biochem. Biophys. 1993; 300: 169-177Crossref PubMed Scopus (50) Google Scholar). This system can generate hundreds of milligrams of E. coli PBGS (up to 30% of the soluble protein). The reasons for high level expression are not fully understood. In this case, the hemB gene is apparently downstream from a naturally strong E. coli promoter, and other poorly understood aspects of gene structure may contribute to the phenomenal levels of constitutive expression observed. A second consideration in artificial gene design is codon usage. The humanALAD gene was analyzed and found to contain clusters of codons that are rarely used by E. coli (see Fig.2 A). In contrast, the E. coli hemB gene contains only one rare codon. Kane (16.Kane J.F. Curr. Opin. Biotechnol. 1995; 6: 494-500Crossref PubMed Scopus (594) Google Scholar) has described how clusters of six specific rare codons can be detrimental to both the quality and quantity of heterologous proteins expressed in E. coli, and specific translational errors have been documented for such clusters (17.Forman M.D. Stack R.F. Masters P.S. Hauer C.R. Baxter S.M. Protein Sci. 1998; 7: 500-503Crossref PubMed Scopus (45) Google Scholar). Hence, in the design of an artificial gene for human PBGS, theE. coli hemB gene structure was mimicked to the greatest extent possible, and rare codons were replaced. Here we describe the design and synthesis of a PCR product (EJhum) containing an artificial gene coding for human PBGS.EJhum mimics a pUC119 version of pCR261 (14.Roessner C.A. Spencer J.B. Ozaki S. Min C. Atshaves B.P. Nayar P. Anousis N. Stolowich N.J. Holderman M.T. Scott A.I. Protein Expression Purif. 1995; 6: 155-163Crossref PubMed Scopus (29) Google Scholar), which gives high level constitutive expression of the E. coli hemB gene (encoding E. coli PBGS) in a variety of E. colihost strains. Successful large-scale protein production of human PBGS was obtained by expression under control of the T7 polymerase system using a plasmid named pMVhum, which encodes the K59 protein corresponding to the ALAD1 allele inserted into the pET3 vector. We present the expression and purification of human PBGS from this construct and a basic characterization of the two human PBGS proteins K59 and N59, the latter a product of site-directed mutagenesis. We also compare the Zn(II) binding properties of the two proteins and address the ability of Pb(II) to displace the essential Zn(II)." @default.
• W1974616082 created "2016-06-24" @default.
• W1974616082 creator A5007664443 @default.
• W1974616082 creator A5011036334 @default.
• W1974616082 creator A5016069898 @default.
• W1974616082 creator A5034405515 @default.
• W1974616082 creator A5053977807 @default.
• W1974616082 creator A5055179810 @default.
• W1974616082 creator A5069206173 @default.
• W1974616082 creator A5081576631 @default.
• W1974616082 creator A5085408345 @default.
• W1974616082 creator A5086591462 @default.
• W1974616082 date "2000-01-01" @default.
• W1974616082 modified "2023-09-28" @default.
• W1974616082 title "An Artificial Gene for Human Porphobilinogen Synthase Allows Comparison of an Allelic Variation Implicated in Susceptibility to Lead Poisoning" @default.
• W1974616082 cites W1562699433 @default.
• W1974616082 cites W1587278423 @default.
• W1974616082 cites W1593421540 @default.
• W1974616082 cites W1593538558 @default.
• W1974616082 cites W161706433 @default.
• W1974616082 cites W1806485888 @default.
• W1974616082 cites W1975514075 @default.
• W1974616082 cites W1979694688 @default.
• W1974616082 cites W1983181891 @default.
• W1974616082 cites W1984193507 @default.
• W1974616082 cites W1991748250 @default.
• W1974616082 cites W1994827016 @default.
• W1974616082 cites W1996874927 @default.
• W1974616082 cites W1998198488 @default.
• W1974616082 cites W1999730856 @default.
• W1974616082 cites W2000129995 @default.
• W1974616082 cites W2000970507 @default.
• W1974616082 cites W2005191555 @default.
• W1974616082 cites W2008387200 @default.
• W1974616082 cites W2013238066 @default.
• W1974616082 cites W2038216298 @default.
• W1974616082 cites W2039707307 @default.
• W1974616082 cites W2048139771 @default.
• W1974616082 cites W2050917233 @default.
• W1974616082 cites W2056567154 @default.
• W1974616082 cites W2065620978 @default.
• W1974616082 cites W2068117410 @default.
• W1974616082 cites W2068224930 @default.
• W1974616082 cites W2070311991 @default.
• W1974616082 cites W2083786664 @default.
• W1974616082 cites W2086863310 @default.
• W1974616082 cites W2093123211 @default.
• W1974616082 cites W2093435661 @default.
• W1974616082 cites W2115526102 @default.
• W1974616082 cites W2125076649 @default.
• W1974616082 cites W2147937034 @default.
• W1974616082 cites W2164130591 @default.
• W1974616082 cites W2281708637 @default.
• W1974616082 cites W2312900235 @default.
• W1974616082 cites W4245658862 @default.
• W1974616082 cites W998537933 @default.
• W1974616082 doi "https://doi.org/10.1074/jbc.275.4.2619" @default.
• W1974616082 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10644722" @default.
• W1974616082 hasPublicationYear "2000" @default.
• W1974616082 type Work @default.
• W1974616082 sameAs 1974616082 @default.
• W1974616082 citedByCount "47" @default.
• W1974616082 countsByYear W19746160822012 @default.
• W1974616082 countsByYear W19746160822014 @default.
• W1974616082 countsByYear W19746160822015 @default.
• W1974616082 countsByYear W19746160822017 @default.
• W1974616082 countsByYear W19746160822018 @default.
• W1974616082 countsByYear W19746160822019 @default.
• W1974616082 countsByYear W19746160822020 @default.
• W1974616082 countsByYear W19746160822021 @default.
• W1974616082 countsByYear W19746160822022 @default.
• W1974616082 countsByYear W19746160822023 @default.
• W1974616082 crossrefType "journal-article" @default.
• W1974616082 hasAuthorship W1974616082A5007664443 @default.
• W1974616082 hasAuthorship W1974616082A5011036334 @default.
• W1974616082 hasAuthorship W1974616082A5016069898 @default.
• W1974616082 hasAuthorship W1974616082A5034405515 @default.
• W1974616082 hasAuthorship W1974616082A5053977807 @default.
• W1974616082 hasAuthorship W1974616082A5055179810 @default.
• W1974616082 hasAuthorship W1974616082A5069206173 @default.
• W1974616082 hasAuthorship W1974616082A5081576631 @default.
• W1974616082 hasAuthorship W1974616082A5085408345 @default.
• W1974616082 hasAuthorship W1974616082A5086591462 @default.
• W1974616082 hasBestOaLocation W19746160821 @default.
• W1974616082 hasConcept C104317684 @default.
• W1974616082 hasConcept C118552586 @default.
• W1974616082 hasConcept C180754005 @default.
• W1974616082 hasConcept C181199279 @default.
• W1974616082 hasConcept C2776682811 @default.
• W1974616082 hasConcept C2781037878 @default.
• W1974616082 hasConcept C2781374117 @default.
• W1974616082 hasConcept C44538786 @default.
• W1974616082 hasConcept C54355233 @default.
• W1974616082 hasConcept C55493867 @default.
• W1974616082 hasConcept C71924100 @default.
• W1974616082 hasConcept C86803240 @default.
• W1974616082 hasConceptScore W1974616082C104317684 @default.
• W1974616082 hasConceptScore W1974616082C118552586 @default.

• next