Carboxyl lyases

Attention has been drawn to the potential of phosphoric acid anhydrides of nucleoside 5 -carboxylic acids (14) as specific reagents for investigating the binding sites of enzymes. For example, (14 B = adenosine) inactivates adenylosuccinate lyase from E. coli almost completely, but has little effect on rabbit muscle AMP deaminase. The rate of hydrolysis of (14) is considerably faster than that of acetyl phosphate, suggesting intramolecular assistance by the 3 -hydroxyl group or the 3-nitrogen atom. 

Hughes MA, Ml Baggs, J al-Dulayymi, MS Baird, PA Williams (2002) Accumulation of 2-aminophenoxa-zine-3-one-7-carboxylate during growth of Pseudomonas putida TW3 on 4-nitro-substituted substrates requires 4-hydroxylaminbenzoate lyase (PnbB). Appl Environ Microbiol 68 4965-4970. 

The activity of exopectate lyase of Clostridium multifermentans depends on the presence of bivalent cations of Ca, Ba, Sr, Mg, and Mn. The activation constant with calcium chloride is 0.06 mM. In a later stage of substrate degradation, an inhibition by Ca2+ was observed at concentrations above 0.5 mM this was attributed by Macmillan and Vaughn105 to a binding of calcium to the carboxyl group of two different molecules of substrate and to the inability of exopectate lyase to split the substrate as this barrier is approached. 

Bacterial enzymes have been reported to catalyze the hydrolysis of nitriles [118][121], A nitrilase (EC 3.5.5.1) acts to hydrolyze aromatic nitriles directly to the carboxylic acid. A nitrile hydratase (a lyase, EC 4.2. E84) acts on short-chain aliphatic nitriles to form the amide. As discussed below, the hydrolysis of nitriles to amides is also documented in mammals, but little appears known about the enzymes involved. 

The possibility that many organic compounds could potentially be precursors of ethylene was raised, but direct evidence that in apple fruit tissue ethylene derives only from carbons of methionine was provided by Lieberman and was confirmed for other plant species. The pathway of ethylene biosynthesis has been well characterized during the last three decades. The major breakthrough came from the work of Yang and Hoffman, who established 5-adenosyl-L-methionine (SAM) as the precursor of ethylene in higher plants. The key enzyme in ethylene biosynthesis 1-aminocyclopropane-l-carboxylate synthase (S-adenosyl-L-methionine methylthioadenosine lyase, EC 4.4.1.14 ACS) catalyzes the conversion of SAM to 1-aminocyclopropane-l-carboxylic acid (ACC) and then ACC is converted to ethylene by 1-aminocyclopropane-l-carboxylate oxidase (ACO) (Scheme 1). 

PLP-dependent enzymes catalyze the following types of reactions (1) loss of the ce-hydrogen as a proton, resulting in racemization (example alanine racemase), cyclization (example aminocyclopropane carboxylate synthase), or j8-elimation/replacement (example serine dehydratase) (2) loss of the a-carboxylate as carbon dioxide (example glutamate decarboxylase) (3) removal/replacement of a group by aldol cleavage (example threonine aldolase and (4) action via ketimine intermediates (example selenocysteine lyase). 

Ammonia lyases catalyze the enantioselective addition of ammonia to an activated double bond. A one-pot, three-step protocol was developed for the enantioselective synthesis of L-arylalanines 50 using phenylalanine ammonia lyase (PAL) in the key step (Scheme 2.20). After formation of the unsaturated esters 48 in situ via a Wittig reaction from the corresponding aldehydes, addition of porcine Ever esterase and basification of the reaction mixture resulted in hydrolysis to the carboxylic acids 49. Once this reaction had gone to completion, introduction of PAL and further addition of ammonia generated the amino acids 50 in good yield and excellent optical purity [22]. 

Benzoylformate decarboxylase (BFD EC 4.1.1.7) belongs to the class of thiamine diphosphate (ThDP)-dependent enzymes. ThDP is the cofactor for a large number of enzymes, including pyruvate decarboxylase (PDC), benzaldehyde lyase (BAL), cyclohexane-1,2-dione hydrolase (CDH), acetohydroxyacid synthase (AHAS), and (lR,6] )-2-succinyl-6-hydroxy-2,4-cyclohexadiene-l-carboxylate synthase (SHCHC), which all catalyze the cleavage and formation of C-C bonds [1]. The underlying catalytic mechanism is summarized elsewhere [2] (see also Chapter 2.2.3). 

Scheme 233 Formation of aliphatic flavour aldehydes and alcohols, a Biotechnological reaction sequence mimicking plant biosynthesis of C6 compounds (green notes ), b HomologoiK reaction sequence in fimgi leading to mushroom-like C8 compounds. The stoichiometric formation of w-oxo-carboxylic acids during hydroperoxide lyase cleavage is not depicted...

ASA-lyase deficiency Pyruvate carboxylase deficiency LPI Pyrroline-5-carboxylate synthase deficiency... 

The first unique enzyme of the important glyoxy-late pathway (Chapter 17), isocitrate lyase, cleaves isocitrate to succinate and glyoxylate (Eq. 13-41).228 The carboxylate group that acts as electron acceptor would presumably be protonated by the enzyme. 

The first data about the bioconversion of farnesol date back to the sixties its degradation pathway is similar to that of geraniol and nerol. Seubert [139] showed that the degradation of farnesol by Pseudomonas citronellolis proceeds through the oxidation of C-l to give famesic acid, followed by carboxylation of the -methyl group. Subsequently, the 2,3-double bond of the dicarboxylic acid is hydrated to a 3-hydroxy acid which is then acted upon by a lyase resulting in the formation of a /Tketo acid and acetic acid. The /Tketo acid readily enters the fatty acid oxidation pathway [29]. 

The majority of useful lyase families utilize anionically functionalized substrates such as pyruvate or dihydroxyacetone phosphate which remain unaltered during catalysis. The charged group thereby introduced into the products (phosphate, carboxylate) not only constitutes a handle for binding of the substrates by the enzymes but also can facilitate the preparative isolation from... 

The degradation of nicotinic acid by Clostridium barkeri involves the cleavage of the intermediate 2,3-dimethylmalate 132 from which propionic and pyruvic acids are formed by a specific lyase (EC 4.1.3.32). In the reverse direction, the enzyme must have the unusual capacity to deprotonate propionic acid at the a-carbon instead of the carboxylic acid function, or next to an anionic car-boxylate. Purified dimethylmalic acid aldolase has been used to catalyze the stereospecific addition of 133 to the oxoacid acceptor, yielding the (2R,3S) configurated dimethylmalic acid 132 at the multi-gram scale [381]. The substrate tolerance of this enzyme has not yet been determined. 

Three modifications of the conventional oxidative citric acid cycle are needed, which substitute irreversible enzyme steps. Succinate dehydrogenase is replaced by fumarate reductase, 2-oxoglutarate dehydrogenase by ferredoxin-dependent 2-oxoglutarate oxidoreductase (2-oxoglutarate synthase), and citrate synthase by ATP-citrate lyase [3, 16] it should be noted that the carboxylases of the cycle catalyze the reductive carboxylation reactions. There are variants of the ATP-driven cleavage of citrate as well as of isocitrate formation [7]. The reductive citric acid... 

The pathway can be divided into two metabolic cycles (Figure 3.4). In the first cycle, acetyl-CoA is carboxylated to malonyl-CoA, which is subsequently reduced and converted into propionyl-CoA via 3-hydroxypropionate as a free intermediate. Propionyl-CoA is carboxylated to methylmalonyl-CoA, which is subsequently converted to succinyl-CoA the latter is then used to activate L-malate by succinyl-CoA L-malate coenzyme A transferase, which forms L-malyl-CoA and succinate. Succinate is oxidized to L-malate via conventional steps. L-Malyl-CoA, the second characteristic intermediate of this cycle, is cleaved by L-malyl-CoA/P-methylmalyl-CoA lyase, thus regenerating the starting molecule acetyl-CoA and releasing gly-oxylate as a first carbon-fixation product [27]. 

Free amino acids are further catabolized into several volatile flavor compounds. However, the pathways involved are not fully known. A detailed summary of the various studies on the role of the catabolism of amino acids in cheese flavor development was published by Curtin and McSweeney (2004). Two major pathways have been suggested (1) aminotransferase or lyase activity and (2) deamination or decarboxylation. Aminotransferase activity results in the formation of a-ketoacids and glutamic acid. The a-ketoacids are further degraded to flavor compounds such as hydroxy acids, aldehydes, and carboxylic acids. a-Ketoacids from methionine, branched-chain amino acids (leucine, isoleucine, and valine), or aromatic amino acids (phenylalanine, tyrosine, and tryptophan) serve as the precursors to volatile flavor compounds (Yvon and Rijnen, 2001). Volatile sulfur compounds are primarily formed from methionine. Methanethiol, which at low concentrations, contributes to the characteristic flavor of Cheddar cheese, is formed from the catabolism of methionine (Curtin and McSweeney, 2004 Weimer et al., 1999). Furthermore, bacterial lyases also metabolize methionine to a-ketobutyrate, methanethiol, and ammonia (Tanaka et al., 1985). On catabolism by aminotransferase, aromatic amino acids yield volatile flavor compounds such as benzalde-hyde, phenylacetate, phenylethanol, phenyllactate, etc. Deamination reactions also result in a-ketoacids and ammonia, which add to the flavor of... 

In addition to resolution approaches, there are three main methods to prepare amino acids by biological methods addition of ammonia to an unsaturated carboxylic acid the conversion of an a-keto acid to an amino acid by transamination from another amino acid, and the reductive animation of an a-keto acid. These approaches are discussed in Chapter 19 and will not be discussed here to avoid duplication. The use of a lyase to prepare L-aspartic acid is included in this chapter as is the use of decarboxylases to access D-glutamic acid. 

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