An artificial gene for human porphobilinogen synthase allows comparison ofan allelic variation implicated in susceptibility to lead poisoning

Porphobilinogen synthase (PBGS) is an ancient enzyme essential to tetrapyrr ole biosynthesis (e.g. heme, chlorophyll, and vitamin B-12). Two common all eles encoding human PBGS, K59 and N59, have been correlated with differenti al susceptibility of humans to lead poisoning. However, a model for human P BGS based on homologous crystal structures shows the location of the alleli c variation to be distant from the active site with its two Zn(II), Previou s 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 homo logous Escherichia coli hemB gene and to remove rare codons that can confou nd heterologous protein expression in E, coli, We have expressed and purifi ed recombinant human PBGS variants K59 and N59 in 100-mg quantities. Both h uman PBGS proteins purified with eight Zn(II)/octamer; Zn(II) binding was s hown to be pH-dependent; and Pb(II) could displace some of the Zn(II), Howe ver, there was no differential displacement of Zn(II) by Pb(II) between K59 and N59, and simple Pb(II) inhibition studies revealed no allelic differen ce.