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Originally published In Press as doi:10.1074/jbc.M100329200 on March 15, 2001

J. Biol. Chem., Vol. 276, Issue 22, 19141-19149, June 1, 2001
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Circular Permutation of 5-Aminolevulinate Synthase
MAPPING THE POLYPEPTIDE CHAIN TO ITS FUNCTION*

Anton V. CheltsovDagger §, Michael J. BarberDagger , and Gloria C. FerreiraDagger ||**

From the Dagger  Department of Biochemistry and Molecular Biology, College of Medicine,  Institute for Biomolecular Science, and || H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, Florida 33612

5-Aminolevulinate synthase is the first enzyme of the heme biosynthetic pathway in non-plant eukaryotes and some prokaryotes. The enzyme functions as a homodimer and requires pyridoxal 5'-phosphate as a cofactor. Although the roles of defined amino acids in the active site and catalytic mechanism have been recently explored using site-directed mutagenesis, much less is known about the role of the 5-aminolevulinate synthase polypeptide chain arrangement in folding, structure, and ultimately, function. To assess the importance of the continuity of the polypeptide chain, circularly permuted 5-aminolevulinate synthase variants were constructed through either rational design or screening of an engineered random library. One percent of the random library clones were active, and a total of 21 active variants had sequences different from that of the wild type 5-aminolevulinate synthase. Out of these 21 variants, 9 displayed unique circular permutations of the 5-aminolevulinate synthase polypeptide chain. The new termini of the active variants disrupted secondary structure elements and loop regions and fell in 100 amino acid regions from each terminus. This indicates that the natural continuity of the 5-aminolevulinate synthase polypeptide chain and the sequential arrangement of the secondary structure elements are not requirements for proper folding, binding of the cofactor, or assembly of the two subunits. Furthermore, the order of two identified functional elements (i.e. the catalytic and the glycine-binding domains) is apparently irrelevant for proper functioning of the enzyme. Although the wild type 5-aminolevulinate synthase and the circularly permuted variants appear to have similar, predicted overall tertiary structures, they exhibit differences in the arrangement of the secondary structure elements and in the cofactor-binding site environment. Taken together, the data lead us to propose that the 5-aminolevulinate synthase overall structure can be reached through multiple or alternative folding pathways.

* This work was supported in part by National Institutes of Health Grant DK52053 (to G. C. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Recipient of American Heart Association/Florida Division Predoctoral Fellowship 9850021FL.

** To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, College of Medicine, University of South Florida, 12901 Bruce B. Downs Blvd., Tampa, FL 33612. Tel.: 813-974-5797; Fax: 813-974-0504; E-mail: gferreir@hsc.usf.edu.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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