title: An artificial gene for human porphobilinogen synthase allows comparison of an allelic variation implicated in susceptibility to lead poisoning.
abstract: Porphobilinogen synthase (PBGS) is an ancient enzyme essential to tetrapyrrole biosynthesis (e.g. heme, chlorophyll, and vitamin B(12)). 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.
authors.affiliation: Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA. EK_Jaffe@fcc.edu
authors.forename: E K
authors.initials: EK
authors.lastname: Jaffe
authors.forename: M
authors.initials: M
authors.lastname: Volin
authors.forename: C R
authors.initials: CR
authors.lastname: Bronson-Mullins
authors.forename: R L
authors.initials: RL
authors.lastname: Dunbrack
authors.forename: J
authors.initials: J
authors.lastname: Kervinen
authors.forename: J
authors.initials: J
authors.lastname: Martins
authors.forename: J F
authors.initials: JF
authors.lastname: Quinlan
authors.forename: M H
authors.initials: MH
authors.lastname: Sazinsky
authors.forename: E M
authors.initials: EM
authors.lastname: Steinhouse
authors.forename: A T
authors.initials: AT
authors.lastname: Yeung
mesh_terms: D000483:Alleles; D001483:Base Sequence; D001667:Binding, Competitive; D018076:DNA, Complementary; D005813:Genes, Synthetic; D020022:Genetic Predisposition to Disease; D014644:Genetic Variation; D006801:Humans; D007855:Lead Poisoning / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D000623:Porphobilinogen Synthase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D015032:Zinc / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D011994:Recombinant Proteins; D000623:Porphobilinogen Synthase; D015032:Zinc