Aminolevulinate synthase: functionally important residues at a glycine loop, a putative pyridoxal phosphate cofactor-binding site.

5-Aminolevulinate synthase catalyzes the first step of the heme biosynthetic pathway in nonplant higher eukaryotes. The enzyme functions as a homodimer and requires pyridoxal 5'-phosphate as its cofactor. Lysine-313 in murine erythroid aminolevulinate synthase has been identified as the residue involved in the Schiff base linkage with pyridoxal 5'-phosphate [Ferreira, G. C., Neame, P. J., & Dailey, H. A. (1993) Protein Sci. 2, 1959-1965]. However, other residues involved in binding and orienting the cofactor remain unknown. We studied the informational content of each residue within an 11 amino acid glycine-rich region, which we propose to be part of the phosphate-binding motif, based on amino acid sequence comparison with other pyridoxal 5'-phosphate-dependent enzymes and nucleotide-binding proteins. Partial random mutagenesis of this region in murine erythroid aminolevulinate synthase gene was followed by an efficient biological selection, using a hemA- Escherichia coli strain to recover functional unnatural enzymes. Among the total of 5444 variants produced, 283 were found to be functional. DNA sequencing results of 226 functional mutants indicated that most residues in this region contained a low informational content, being able to tolerate several other amino acid substitutions. However, three residues, namely, Arg-149, Gly-142, and Gly-144, were found to contain high informational content; Arg-149 was conserved in all of the functional mutants sequenced, while Gly-142 and Gly-144 could only tolerate alanine replacement. Two codon-specific random libraries of Arg-149, and Gly-142 and -144, respectively, were constructed to test further the stringency of these three positions.(ABSTRACT TRUNCATED AT 250 WORDS)