Random mutagenesis of 1-aminocyclopropane-1-carboxylate synthase: a key enzyme in ethylene biosynthesis

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 95; no. 17; pp. 9796 - 9801
• Main Authors: Tarun, A.S. (Plant Gene Expression Center, Albany, CA.), Lee, J.S, Theologis, A
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 18.08.1998; National Acad Sciences; National Academy of Sciences
• Subjects: ACC SYNTHASE; ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; ADN; Amino Acid Sequence; AMINO ACID SEQUENCES; Amino acids; Base Sequence; Binding Sites - genetics; Biochemistry; Biological Sciences; CHEMICAL COMPOSITION; COMPLEMENTARY DNA; COMPLEMENTATION; COMPOSICION QUIMICA; COMPOSITION CHIMIQUE; DNA; DNA Primers - genetics; Enzymes; ENZYMIC ACTIVITY; ESCHERICHIA COLI; Escherichia coli - enzymology; Escherichia coli - genetics; Ethylenes - biosynthesis; Flowers & plants; GENE; Gene Expression; GENES; Genetic Complementation Test; Genetic mutation; Genetic screening; GENETICA; GENETICS; GENETIQUE; INDUCED MUTATION; Isoenzymes - genetics; Isoenzymes - metabolism; ISOLEUCINE AUXOTROPHS; LIASAS; LYASE; LYASES; Lyases - genetics; Lyases - metabolism; LYCOPERSICON ESCULENTUM; Lycopersicon esculentum - enzymology; Lycopersicon esculentum - genetics; MISSENSE MUTATIONS; Molecular Sequence Data; MUTACION; MUTACION INDUCIDA; Mutagenesis; Mutagenesis, Site-Directed; MUTANT; MUTANTES; MUTANTS; MUTATION; MUTATION PROVOQUEE; PCR; Plasmids; POLYMERASE CHAIN REACTION; Protein synthesis; Pseudomonas; Pseudomonas - enzymology; Pseudomonas - genetics; TARGETED MUTAGENESIS
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.95.17.9796

1-Aminocyclopropane-1-carboxylate synthase (ACC synthase, EC 4.4.1.14) catalyzes the rate-limiting step in the ethylene biosynthetic pathway in plants. To determine the amino acid residues critical for the structure and function of this enzyme, the tomato Le-ACS2 isoenzyme has been subjected to both site-directed and PCR random mutagenesis. Mutant ACC synthases with reduced enzyme activity have been selected by using a genetic screen based on the functional complementation of an Escherichia coli Ile auxotroph that has been engineered to express ACC deaminase from Pseudomonas sp. The DNA sequence of almost 1,000 clones has been determined, and 334 single missense mutations have been selected for analysis. We have identified three classes of mutants based on their activity and expression in E. coli. Class I and II mutants have the same level of protein expression as the wild type, but their enzyme activity is reduced to 0-5% and 5-50%, respectively. Class III mutants have neither activity nor detectable protein expression. The inactive mutations are clustered in regions that are highly conserved among various ACC synthases. This library of mutants will facilitate the elucidation of structure-function relationships of this regulatory enzyme