Identification and functional differentiation of two type I fatty acid synthases in Brevibacterium ammoniagenes.
The fatty acid synthase (FAS) from Brevibacterium ammoniagenes is a homohexameric multienzyme complex that catalyzes the synthesis of both saturated and unsaturated fatty acids. By immunological screening of a B. ammoniagenes expression library, an fas DNA fragment was isolated and subsequently used to clone the entire gene together with its flanking sequences. Within 10,525 bp of sequenced DNA, the 9,189-bp FAS coding region was identified, corresponding to a protein of 3,063 amino acids with a molecular mass of 324,910 Da. This gene (fasA) encodes, at its 5' end, the same amino acid sequence as is observed with purified B. ammoniagenes FAS. A second reading frame encoding another B. ammoniagenes FAS variant (FasB) had been identified previously. Both sequences are colinear and exhibit 61 and 47% identity at the DNA and protein levels, respectively. By using specific antibodies raised against a unique peptide sequence of FasB, this enzyme was shown to represent only 5 to 10% of the cellular FAS protein. Insertional inactivation of the FasB coding sequence causes no defective phenotype, while fasA disruptants require oleic acid for growth. Correspondingly, oleate-dependent B. ammoniagenes cells obtained by ethyl methanesulfonate mutagenesis were complemented by transformation with fasA DNA but not with fasB DNA. The data indicate that B. ammoniagenes contains two related though differently expressed type I FASs. FasA represents the bulk of cellular FAS protein and catalyzes the synthesis of both saturated and unsaturated fatty acids, while the minor variant, FasB, cannot catalyze the synthesis of oleic acid.
Stuible
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Lehrstuhl für Biochemie der Universität Erlangen-Nürnberg, Germany.
Wagner
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C
Andreou
I
I
Huter
G
G
Haselmann
J
J
Schweizer
E
E
D018830:Adhesins, Escherichia coli / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D000954:Antigens, Surface / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D001951:Brevibacterium / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli; D064429:Fatty Acid Synthases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D036781:Fimbriae Proteins; D015870:Gene Expression; D015723:Gene Library; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D014644:Genetic Variation; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D019301:Oleic Acid; D009829:Oleic Acids / Q000378:metabolism; D010957:Plasmids; D016364:Reading Frames; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid
D018830:Adhesins, Escherichia coli; D000942:Antigens, Bacterial; D000954:Antigens, Surface; D007527:Isoenzymes; D009829:Oleic Acids; C035391:antigen 987P, E coli; D036781:Fimbriae Proteins; D019301:Oleic Acid; D064429:Fatty Acid Synthases