Broad Substrate Stereospecificity of the Mycobacterium tuberculosis 7-Keto-8-aminopelargonic Acid Synthase

Abstract

Biotin is an essential enzyme cofactor required for carboxylation and transcarboxylation reactions. The absence of the biotin biosynthesis pathway in humans suggests that it can be an attractive target for the development of novel drugs against a number of pathogens. 7-Keto-8-aminopelargonic acid (KAPA) synthase (EC 2.3.1.47), the enzyme catalyzing the first committed step in the biotin biosynthesis pathway, is believed to exhibit high substrate stereospecificity. Biotin (vitamin H) is an essential cofactor required for a number of carboxylation, transcarboxylation, and decarboxylation reactions in all living organisms. Its participation in gluconeogenesis, metabolism of amino acids, and initiation of fatty acid biosynthesis makes it an indispensable entity in cellular metabolism. However, only plants and microorganisms are capable of synthesizing biotin, whereas mammals obtain it either from the diet or from the intestinal microflora (1). The biotin biosynthesis pathway has been studied in detail in microbes like Escherichia coli, Bacillus subtilis, Bacillus sphaericus, and Saccharomyces cerevisiae and in plants like Arabidopsis thaliana and Lavandula vera. With the exception of the first step, which is the synthesis of pimeloyl-CoA, the remaining four steps of the pathway (Scheme 1) are invariant in all biotin-synthesizing organisms and are carried out by four committed enzymes (2). The first of these four steps is the decarboxylative condensation of pimeloyl-CoA and L-alanine to 7-keto-8-aminopelargonic acid (KAPA) 3 catalyzed by KAPA synthase, followed by the DAPA synthase-catalyzed transfer of an amino group from S-adenosylmethionine to KAPA, forming DAPA. DAPA is then converted to dethiobiotin by the addition of CO 2 in an ATP-dependent reaction catalyzed by dethiobiotin synthetase, and finally the biotin synthase-catalyzed insertion of a sulfur atom between the reactive methyl and methylene carbon atoms adjacent to the ureido ring of dethiobiotin to generate biotin (1-3).KAPA synthase is a PLP-dependent enzyme belonging to subclass II of the aminotransferase family. Along with 5-aminolevulinate synthase, serine palmitoyltransferase, and 2-amino-3-oxobutyrate-CoA ligase, which typically catalyze the Claisen condensations between amino acids and acyl-CoA thioesters, it forms the ␣-oxoamine synthase subfamily. The similarities in the sequences of these enzymes as well as the reactions catalyzed by them indicate that they share a common catalytic mechanism (4 -6). Availability of the three-dimensional structures of these enzymes has provided a structural basis for understanding the reactions catalyzed by them (4, 7-9). The knowledge of the reaction mechanism of KAPA synthase in particular is based on studies on the Bacillus sphaericus and E. coli enzymes (7, 10 -11). It involves the displacement of lysine from the internal aldimine complex with PLP resulting in the formation of the external aldimine between PLP and L-alanine. This is followed by the abstraction of the C␣-H proton of the L-alanine external ald...

Aminolaevulinic acid synthase of Rhodobacter capsulatus: high-resolution kinetic investigation of the structural basis for substrate binding and catalysis

“…Thus we monitored the formation of the quinonoid intermediate I in the R. capsulatus ALAS-catalysed reaction using the substrate analogue O-methylglycine and transient kinetics under presteady-state conditions and following the changes in absorbance at 510 nm ( Figure 3 and Table 1). The observation of the quinonoid intermediate I in the R. capsulatus ALAS-catalysed reaction is in good agreement with similar results obtained from experiments performed with E. coli and Mycobacterium tuberculosis AONS [16,31] and SPT [32]. Similar to the reaction of the ALASglycine complex with succinyl-CoA, the kinetic trace for the reaction of the ALAS-O-methylglycine complex with succinyl-CoA was best described as a four-step process [eqn (1); k 1 = 2.96 s − 1 , k 2 = 0.19 s − 1 , k 3 = 32.10 s − 1 and k 4 = 0.64 s − 1 ; Table 1).…”

Molybdenum enzymes and molybdenum cofactor in mycobacteria

“…Research has discovered the complete biotin biosynthesis pathways in many different microbes, but the study on the biotin biosynthesis in M. tuberculosis is exiguous. Although the biotin synthesis genes in M. tuberculosis were deciphered [Cole et al, 1998;Camus et al, 2002] and some products of these genes were investigated [Bhor et al, 2006;Mann and Ploux, 2006], the process of biotin biosynthesis remains an unknown quantity.Biotin is unstable and spontaneously oxidizes to biotin sulfoxide. The Escherichia coli biotin d-sulfoxide reductase reduces biotin sulfoxide to biotin [del Campillo-Campbell and Campbell, 1982] and has essential role in assimilation of free oxidized methionines in E. coli [Ezraty et al, 2005].…”

The First Thermophilic α-Oxoamine Synthase Family Enzyme That Has Activities of 2-Amino-3-ketobutyrate CoA Ligase and 7-Keto-8-aminopelargonic Acid Synthase: Cloning and Overexpression of the Gene from an Extreme Thermophile,Thermus thermophilus, and Characterization of Its Gene Product

“…Recently, it was reported that Mycobacterium tuberculosis KAPA synthase shows a unique substrate stereospecificity: it uses both L-and D-alanine, 37) but the recombinant TTHA1582 protein did not utilize Dalanine.…”