Tracking the Evolution of Porphobilinogen Synthase Metal Dependence in Vitro Frederic Fre `re 1 , Heike Reents 1 , Wolf-Dieter Schubert 2 , Dirk W. Heinz 2 and Dieter Jahn 1 * 1 Institute of Microbiology Technical University Braunschweig Spielmannstrasse 7, D-38106 Braunschweig, Germany 2 Divison of Structural Biology German Research Center for Biotechnology, Mascheroder Weg 1, D-38124 Braunschweig Germany Metal ions are indispensable cofactors for chemical catalysis by a plethora of enzymes. Porphobilinogen synthases (PBGSs), which catalyse the second step of tetrapyrrole biosynthesis, are grouped according to their dependence on Zn 2C . Using site-directed mutagenesis, we embarked on transforming Zn 2C -independent Pseudomonas aeruginosa PBGS into a Zn 2C -dependent enzyme. Nine PBGS variants were generated by permutationally introducing three cysteine residues and a further two residues into the active site of the enzyme to match the homologous Zn 2C - containing PBGS from Escherichia coli. Crystal structures of seven enzyme variants were solved to elucidate the nature of Zn 2C coordination at high resolution. The three single-cysteine variants were invariably found to be enzymatically inactive and only one (D139C) was found to bind detectable amounts of Zn 2C . The double mutant A129C/D139C is enzymatically active and binds Zn 2C in a tetrahedral coordination. Structurally and functionally it mimics mycobacterial PBGS, which bears an equivalent Zn 2C -coordination site. The remaining two double mutants, without known natural equivalents, reveal strongly distorted tetrahedral Zn 2C - binding sites. Variant A129C/D131C possesses weak PBGS activity while D131C/D139C is inactive. The triple mutant A129C/D131C/D139C, finally, displays an almost ideal tetrahedral Zn 2C -binding geometry and a significant Zn 2C -dependent enzymatic activity. Two additional amino acid exchanges further optimize the active site architecture towards the E. coli enzyme with an additional increase in activity. Our study delineates the potential evolutionary path between Zn 2C -free and Zn 2C -dependent PBGS enyzmes showing that the rigid backbone of PBGS enzymes is an ideal framework to create or eliminate metal dependence through a limited number of amino acid exchanges. q 2004 Elsevier Ltd. All rights reserved. Keywords: crystal structure; evolution; metalloenzyme; porphobilinogen synthase; Pseudomonas aeruginosa *Corresponding author Introduction Porphobilinogen synthase (PBGS) is an essential constituent of the tetrapyrrole biosynthetic appa- ratus, present in the majority of known organisms. It catalyses the second step of this pathway, the asymmetric condensation of two molecules of 5-aminolevulinic acid (ALA) to yield the mono- pyrrolic product porphobilinogen (PBG) in a Knorr- type reaction 1–5 (Figure 1). The active form of PBGS is a homo-octamer, as documented by crystal struc- tures from Saccharomyces cerevisiae (ScPBGS), Pseudomonas aeruginosa (PaPBGS), Escherichia coli (EcPBGS) 2,6–8 and Homo sapiens (PDB entry 1E51 and Breinig et al. 9 ). Each monomer consists of a 0022-2836/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. Abbreviations used: ALA, 5 aminolevulinic acid; PBG, porphobilinogen; PBGS, porphobilinogen synthase; b-ME, b-mercaptoethanol; PaPBGS, PBGS from Pseudomonas aeruginosa; EcPBGS, PBGS from E. coli; ScPBGS, PBGS from Saccharomyces cerevisiae; PaPBGS mutants: CXX, A129C; XCX, D131C; XXC, D139C; CCX, A129C/D131C; CXC, A129C/D139C; XCC, D131C/D139C; CCC, A129/D131C/D139C; CCCE, A129C/D131C/D139C/P132E; CCCER, A129C/D131C/D139C/P132E/K229R. E-mail address of the corresponding author: d.jahn@tu-bs.de doi:10.1016/j.jmb.2004.10.053 J. Mol. Biol. (2005) 345, 1059–1070