Synthesis of 7-Oxo-8-Aminopelargonic Acid, a Biotin Vitamer, in Cell-free Extracts of Escherichia coli Biotin Auxotrophs

Abstract

The enzymatic synthesis of 7-oxo-8-aminopelargonic acid (7-KAP) from pimelylcoenzyme A and L-alanine was demonstrated in cell-free extracts of a biotin mutant of Escherichia coli K-12 which excretes only 7-KAP into the growth medium. This biotin vitamer was identified by its chromatographic and electrophoretic properties. The enzyme (7-KAP synthetase) was repressed when the organism was grown in biotin concentrations greater than 0.2 ng/ml. The parent strain and members of other mutant groups that excrete 7-KAP, in addition to other vitamers, also exhibited synthetase activity. A mutant group that failed to excrete 7-KAP was further subdivided into three groups, one of which lacked synthetase activity. These results are discussed in relation to a previously proposed scheme for biotin biosynthesis in which the formation of 7-KAP is considered the point of entry for pimelic acid into the biotin pathway. Pimelic acid was established as a direct precursor of biotin by the isotopic studies of Eisenberg (3) and H

The 8-amino-7-oxopelargonate synthase from Bacillus sphaericus. Purification and preliminary characterization of the cloned enzyme overproduced in Escherichia coli

“…Pimeloyl-CoA was synthesized as described by Eisenberg & Star [3] starting from pimeloyl chloride rather than from the polymeric anhydride of pimelic acid, the former being readily available, in contrast with the latter. Pimeloyl-CoA was routinely determined as its hydroxamate [8].…”

“…fundamental aspects (enzymology, genetics) as well as industrial microbiological production of biotin, we have investigated the second step of this metabolic pathway in Bacillus sphaericus: the transformation of pimeloyl-CoA into 8-amino-7-oxopelargonate (AOP) (Scheme 1) [1,2]. This interesting reaction is catalysed by AOP synthase [6-carboxyhexanoyl-CoA: L-alanine carboxyhexanoyltransferase (decarboxylating); EC 2.3.1.47], which has been detected in the cell-free extract of different bacteria [3,4], and partially purified from B. sphaericus by Izumi and colleagues [5]. This enzyme belongs to the family of pyridoxal 5'-phosphatedependent enzymes that catalyse the condensation of an acyl-CoA with an amino acid leading to an a-amino ketone [5-aminolaevulinate synthase (EC 2.3.1.37), 2-amino-3-oxobutyrate: CoA ligase (EC 2.3.1.29) and serine palmitoyltransferase (EC 2.3.1.50)].…”

Investigation of the first step of biotin biosynthesis in Bacillus sphaericus. Purification and characterization of the pimeloyl-CoA synthase, and uptake of pimelate

“…The first common intermediate of this pathway, in these two bacteria, is pimeloyl-CoA (Scheme 1). Indeed, the transformation of pimeloyl-CoA to 8-amino-7-oxononanoate has been demonstrated in cell-free extracts of E. coli [3], B. sphaericus [4] and other bacteria [5], and we have recently reported the first purification to homogeneity of the 8-amino-7-oxononanoate synthase (EC 2.3.1.47) [6]. On the other hand, the origin of pimeloyl-CoA is not clear in all the bacteria so far studied.…”

Crystallization and preliminary X-ray study of the 8-amino-7-oxopelargonate synthase fromBacillus sphaericus

“…1). This reaction is catalyzed by 8-amino-7-oxopelargonate synthase (AOPS), a pyridoxal 5'-phosphate (PLP) dependent enzyme that has been detected in various microorganisms (Eisenberg & Star, 1968;Izumi, Morita, Tani & Ogata, 1973;Izumi, Sato, Tani & Ogata, 1973). The first purification to homogeneity of this enzyme cloned from B. sphaericus overproduced in Escherichia coli, and its preliminary characterization has been reported recently (Ploux & Marquet, 1992).…”