Biotin biosynthesis pathway

K Hatakeyama, M Kobayashi, H Yukawa. Analysis of biotin biosynthesis pathway in coryneform bacteria Brevibacterium flavum. Methods Enzymol 279 339-348, 1997. 

The biotin biosynthetic pathway is outlined in Fig. 25 [115]. Pimeloyl-CoA, synthesized by a variation of the fatty acid biosynthesis pathway, is condensed with alanine to give 142. 

The pathway for biotin biosynthesis elucidated in bacteria by application of the mutants technique was proposed by Eisenberg in 1968. ° This pathway, which has never been questioned ever since, is depicted in Figure 2. It has also been shown that the same pathway is present in plants."... 

For several years, she studied chemical reaction mechanisms, especially those involving sulfoxide carbanions and their synthetic applications. Then, she turned to mechanistic enzymology. Her main interests concerned steroid isomerases and cytochrome P-450, vitamin K-dependent carboxylations, and biotin biosynthesis. She contributed to the mechanistic understanding of several enzymes of the pathway, namely, amino-oxopelargo-nate synthase, diaminopelargonate aminotransferase, and more importantly biotin synthase. 

The biosynthesis pathways of the vitamins thiamine (Bl), pyridoxine (B6), and biotin (B7) have been elucidated during the last 10 years to some detail. It became clear that in all cases enzymes catalyzing unusual or complex biochemical reaction mechanisms are involved, which perform at least in vitro with very low catalytic efficiency. Previous attempts to breed B. suhtilis production strains for these vitamins as a base for the development of superior processes to supersede the decades old chemical processes are reviewed here briefly. These efforts followed the blueprints that were successful for other metabolites like amino acids, nucleotides, the vitamins mentioned above, and others. However, the metabolic fluxes toward the vitamins Bl, B6, and B7 have proven to be particularly adamant to engineering. The strains that were obtained overproduced these value compounds only at marginal levels. 

Rittenberg and Bloch showed in the late 1940s that acetate units are the building blocks of fatty acids. Their work, together with the discovery by Salih Wakil that bicarbonate is required for fatty acid biosynthesis, eventually made clear that this pathway involves synthesis of malonyl-CoA. The carboxylation of acetyl-CoA to form malonyl-CoA is essentially irreversible and is the committed step in the synthesis of fatty acids (Figure 25.2). The reaction is catalyzed by acetyl-CoA carboxylase, which contains a biotin prosthetic group. This carboxylase is the only enzyme of fatty acid synthesis in animals that is not part of the multienzyme complex called fatty acid synthase. 

Biotin 0.15-0.3 mg/day. The discovery that biotin deficiency in young chickens can lead to sudden death resulted in a recommendation to supplement infant formulations with biotin.3 Desthiobiotin, in which the sulfur has been removed and replaced by two hydrogen atoms, can replace biotin in some organisms and appears to lie on one pathway of biosynthesis. b/C Oxybiotin, in which the sulfur has been replaced by oxygen, is active for many organisms and partially active for others. No evidence for conversion to biotin itself has been reported, and oxybiotin may function satisfactorily in at least some enzymes. 

Eight enzyme-catalyzed reactions are involved in the conversion of acetyl-CoA into fatty acids. The first reaction is catalyzed by acetyl-CoA carboxylase and requires ATP. This is the reaction that supplies the energy that drives the biosynthesis of fatty acids. The properties of acetyl-CoA carboxylase are similar to those of pyruvate carboxylase, which is important in the gluconeogenesis pathway (see chapter 12). Both enzymes contain the coenzyme biotin covalently linked to a lysine residue of the protein via its e-amino group. In the last section of this chapter we show that the activity of acetyl-CoA carboxylase plays an important role in the control of fatty acid biosynthesis in animals. Regulation of the first enzyme in a biosynthetic pathway is a strategy widely used in metabolism. 

The pathway The first committed step in fatty acid biosynthesis is the carboxylation of acetyl CoA to form malonyl CoA which is catalyzed by the biotin-containing enzyme acetyl CoA carboxylase. Acetyl CoA and malonyl CoA are then converted into their ACP derivatives. The elongation cycle in fatty acid synthesis involves four reactions condensation of acetyl-ACP and malonyl-ACP to form acetoacetyl-ACP releasing free ACP and C02, then reduction by NADPH to form D-3-hydroxybutyryl-ACP, followed by dehydration to crotonyl-ACP, and finally reduction by NADPH to form butyryl-ACP. Further rounds of elongation add more two-carbon units from malonyl-ACP on to the growing hydrocarbon chain, until the C16 palmitate is formed. Further elongation of fatty acids takes place on the cytosolic surface of the smooth endoplasmic reticulum (SER). 

Although the fatty acid oxidation scheme works neatly for even-numbered chain lengths, it can t work completely for fatty acids that contain an odd number of carbons. P-oxidation of these compounds leads to propionyl-CoA and acetyl-CoA, rather than to two acetyl-CoA at the final step. The propionyl-CoA is not a substrate for the TCA cycle or other simple pathways. Propionyl-CoA undergoes a carboxylation reaction to form methylmalonyl-CoA. This reaction requires biotin as a cofactor, and is similar to an essential step in fatty acid biosynthesis. Methylmalonyl-CoA is then isomerized by an epimerase and then by methylmalonyl-CoA mutase—an enzyme that uses Vitamin Bi2 as a cofactor—to form succinyl-CoA, which is a TCA-cycle intermediate. 

Vitamin B12 is essential for the methylmalonyl-CoAmutase reaction. Methylmalonyl-CoA mutase is required during the degradation of odd-chain fatty acids and of branched-chain amino acids. Odd-chained fatty acids lead to propionyl-CoA as the last step of P-oxida-tion. Methylmalonyl-CoA can be derived from propionyl-CoA by a carboxylase reaction similar to that of fatty acid biosynthesis. The cofactor for this carboxylation reaction is biotin, just as for acetyl-CoA carboxylase. The reaction of methylmalonyl-CoA mutase uses a free radical intermediate to insert the methyl group into the dicar-boxylic acid chain. The product is succinyl-CoA, a Krebs cycle intermediate. The catabolisms of branched-chain lipids and of the branched-chain amino acids also require the methylmalonyl-CoA mutase, because these pathways also generate propionyl-CoA. 

Branching of pathways is relevant in several cases. Thus, intermediates of the porphyrin biosynthetic pathway serve as precursors for chlorophyll (17, Fig. 2) and for the corrinoid ring systems of vitamin B12 (20, Fig. 2) (17). 1-Deoxy-D-xylulose 5-phosphate (43) serves as an intermediate for the biosynthesis of pyridoxal 5 -phosphate (39, Fig. 5), for the terpenoid precursor IPP (86) via the nonmevalonate pathway (Fig. 11), and for the thiazole moiety of thiamine pyrophosphate (46, Fig. 4). 7,8-Dihydroneopterin triphosphate (29, Fig. 3) serves as intermediate in the biosynthetic pathways of tetrahydrofolate (33) and tetrahydrobiopterin (31). The closely related compound 7,8-dihydroneopterin 2, 3 -cyclic phosphate is the precursor of the archaeal cofactor, tetrahydromethanopterin (34) (58). A common pyrimidine-type intermediate (23) serves as precursor for flavin and deazaflavin coenzymes. Various sulfur-containing coenzymes (thiamine (9), lipoic acid (7), biotin (6), Fig. 1) use a pyrosulfide protein precursor that is also used for the biosynthesis of inorganic sulfide as a precursor for iron/sulfur clusters (12). 

Even though E. coli is a very well-studied bacterium, many interesting mechanistic problems in cofactor biosynthesis in this organism remain unsolved. The mechanisms for the formation of the nicotinamide ring of NAD, the pyridine ring of pyridoxal, the pterin system of molybdopterin, and the thiazole and pyrimidine rings of thiamin are unknown. The sulfur transfer chemistry involved in the biosynthesis of lipoic acid, biotin, thiamin and molybdopterin is not yet understood. The formation of the isopentenylpyrophosphate precursor to the prenyl side chain of ubiquinone and menaquinone does not occur by the mevalonate pathway. None of the enzymes involved in this alternative terpene biosynthetic pathway have been characterized. The aim of this review is to focus attention on these unsolved mechanistic problems. 

The a-oxoamine synthases family is a small group of fold-type I enzymes that catalyze Claisen condensations between amino acids and acyl-CoA thioesters (Figure 16). Members of this family are (1) 8-amino-7-oxononanoate (AON) synthase (AONS), which catalyzes the first committed step in the biosynthesis of biotine, (2) 5-aminolevulinate synthase (ALAS), responsible for the condensation between glycine and succinyl-CoA, which yields aminolevulinate, the universal precursor of tetrapyrrolic compounds, (3) serine palmitoyltransferase (SPT), which catalyzes the first reaction in sphingolipids synthesis, and (4) 2-amino-3-ketobutyrate CoA ligase (KBL), involved in the threonine degradation pathway. With the exception of the reaction catalyzed by KLB, all condensation reactions involve a decarboxylase step. 

In contrast to our understanding of the biosynthesis of cofactors, relatively little is known about cofactor degradation. Some previous research has been carried out to identify intermediates on these catabolic pathways, but very little information is available on the genes involved and on the enzymol-ogy. In this chapter we summarize our current understanding of the pyridoxal phosphate, riboflavin, heme, thiamin, biotin, nicotinamide adenine dinucleotide (NAD), folate, lipoate, and coenzyme A catabolic pathways in all life-forms and discuss mechanistic aspects of the most interesting catabolic reactions. 

---