Biotin Metabolism in E. coli K-12
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Biotin Metabolism in E. coli K-12

One of the most fascinating cofactors involved in central pathways of pro- and eukaryotic cell metabolism belongs to the B-Complex group of Vitamins known as Biotin or Vitamin-H. Biotin is a colorless and orthorhombic, consisting of two fused rings: an Imidazol (Ureido) and a Sulfur-containing (Tetrahydrothiophene) ring; and the latter is extended via a Valeric acid side chain, which is attached in a cis-configuration with respect to the Ureido ring. Both rings are fused in cis. Biotin contains three chiral carbon atoms, resulting in eight possible Stereoisomers. However, only the Biotin possesses Vitamin activity. The correct chemical name for the cofactor is: Hexahydro-2-Oxo-1H-Thieno (3,4-D) Imidazole-4-Pentanoic Acid. It is noteworthy that this Vitamin withstands high temperatures; and solutions can be sterilized by autoclaving (Ref.1). However, the molecule is insoluble in most organic solvents. In mammals, Biotin is present only in very small amounts and is supplied by intestinal bacteria. As Biotin biosynthesis is unique to plants and many microorganisms, enzymes of this pathway are potential targets for the development of safe antimicrobial drugs and herbicides. Microbial metabolism of Biotin is highly intriguing and best understood in a handful of microorganisms (Ref.1 & 2).

The nucleotide sequence of the Biotin biosynthetic (bio) Operon in E. coli, a Gram-Negative bacterium, is a 5.8 kb (5.8-Kilobase) region, and contains five Biotin Operon genes, bioA, B, F, C, and D, and an open reading frame of unknown function. In E. coli Biotin is synthesized from Pimeloyl-CoA (Pimeloyl Coenzyme-A). The four steps known to be involved in the conversion of Pimeloyl-CoA to Biotin are highly conserved in both Gram-Positive and Gram-Negative bacteria. Interestingly, the precursors of Pimeloyl-CoA are not known in some of the Gram-Negative bacteria, while the Gram-Positive bacteria utilize Pimelic Acid. However in E.coli K-12, the first step in Biotin biosynthesis involves BioC (Methyltransferase) and BioH (Serine Hydrolase) proteins (product of bioC and bioH gene, respectively) that converts precursors from carbon source to Pimeloyl-CoA. Pimeloyl-CoA is then converted to KAPA (7-Keto-8-Aminopelargonic Acid) by bioF gene product known as AONS (8-Amino-7-Oxononanoate Synthase) or KAPA Synthase or BioF protein. This reaction is also facilitated by Alanine. Formation of KAPA leads to release of Coenzyme-A and Carbondioxide. The antepenultimate step in this pathway, the conversion of KAPA to DAPA (7,8-Diaminopelargonic Acid), is catalyzed by DANS (7,8-Diaminononanoate Synthase) or DAPA Aminotransferase or BioA protein; a product of bioA gene. It is a Vitamin-B6 dependent enzyme and uses S-AdoMet (S-Adenosylmethionine) as Amino-group donor, an unusual feature among Aminotransferases. The next step is conversion of DAPA to Dethiobiotin, catalyzed by bioD gene product DtbS (Dethiobiotin Synthase) or BioD protein. This enzyme catalyzes the ATP (Adenosine Triphosphate)-dependent formation of Dethiobiotin from DAPA and CO2. ATP is converted to ADP (Adenosine Diphosphate) with the release of Pi (Inorganic Phosphate). The final step is chemically most challenging which requires the insertion of a Sulfur atom (from sources like S-AdoMet and Cysteine); between the non-reactive methyl and methylene carbon atoms adjacent to the Ureido ring of Dethiobiotin. In enzymological terms, this reaction of the pathway is least understood (Ref.1 & 2). Compelling evidence proves that bioB gene product Biotin Synthetase or BioB protein (an Iron-Sulfur dimeric enzyme) is essential for this reaction to occur. Although it is widely accepted that the Sulfur originates from a Cysteine, it is not clear whether Cysteine Desulfurase possibly acts as a Desulfurase to supply the required Sulfur from Cysteine, or whether the BioB protein itself is involved in liberating the Sulfur from a free Cysteine (Ref.3).

Again the covalent addition of Biotin to proteins is catalyzed by Biotin-Protein Ligase. For Biotin-Protein Ligase, Biotin addition occurs as an ATP-dependent, two-step reaction that, in the first step, involves synthesis of the intermediate, Biotinyl-5-AMP. In the second step, Biotinyl-5-AMP is used to transfer Biotin, with release of AMP, to a specific Lysine residue in a highly conserved region in Apocarboxylase. Upon addition of Biotin as a co-factor, the Apocarboxylase are converted into active Holocarboxylase. The role of Biotin in Carboxylases is to act as vector for carboxyl-group transfer between donor and acceptor molecules during Carboxylation reaction. Protein-bound Biotin must be released from the Holocarboxylases to which it is attached before it can be used again in Carboxylation reactions and this release is facilitated by Protease and results in the formation of Biocytin (Biotin-Lysine) complex. Hence a complete DNA sequence of the Biotin Operon may facilitate further studies of the structure and function of the bio genes and the enzymes they produce. The sequence information is useful in the study of the evolution of bidirectional transcription in bacteria. The bioA and bioF genes may be evolutionarily related; because the BioF enzyme reaction product is the substrate for the BioA enzyme, both enzymes utilize a PLP (Pyridoxal-5’-Phosphate) cofactor and have comparable molecular weight (Ref.3). The sequence analysis of the bioA and bioF genes has revealed insights into their evolution. Although these genes may have evolved from a common ancestor, they probably diverged before the creation of vertebrates because they share greater homologies to related enzymes in vertebrates than to each other. As Biotin plays a pivotal role in functioning of Biotin-dependent Carboxylases, Fatty Acid Biosynthesis, Gluconeogenesis and Amino Acid Metabolism and since its bioavailability is rather low, consequently, there is the need to produce sufficient amounts of Biotin either by a classic chemical de novo synthesis or by employing biotechnology (Ref.4).