Hydratase, hydration

From substrate specificity studies (23,24), it seems that DS5 hydratase hydrates a specific carbon position of the unsaturated fatty acid substrates. To clarify this point and the effect of substrate carbon chain length on the strain DS5 hydratase activity, we studied the hydration of mono-, di-, and triunsaturated C-18 fatty acids as well as other carbon chain length monounsaturated fatty acids. 

The strain DS5 system produced more keto product from palmitoleic and oleic acids and more hydroxy product from myristoleic, linoleic, and a- and y-linolenic acids. The reason for fliis preference is not clear. Among die 18-carbon unsaturated fatty acids, an additional double bond in either side of die C-10 position lowers the enzyme hydration activity. A hterature search revealed diat all microbial hydratases hydrate oleic and linoleic acids at the C-10 position (Fig. 2). Therefore, die positional specificity of microbial hydratases might be universal. 

There are two distinct classes of enzymes that hydrolyze nitriles. Nittilases (EC 3.5.5. /) hydrolyze nittiles directiy to corresponding acids and ammonia without forming the amide. In fact, amides are not substrates for these enzymes. Nittiles also may be first hydrated by nittile hydratases to yield amides which are then converted to carboxyUc acid with amidases. This is a two-enzyme process, in which enantioselectivity is generally exhibited by the amidase, rather than the hydratase. 

There are two pathways for the degradation of nitriles (a) direct formation of carboxylic acids by the activity of a nitrilase, for example, in Bacillus sp. strain OxB-1 and P. syringae B728a (b) hydration to amides followed by hydrolysis, for example, in P. chlororaphis (Oinuma et al. 2003). The monomer acrylonitrile occurs in wastewater from the production of polyacrylonitrile (PAN), and is hydrolyzed by bacteria to acrylate by the combined activity of a nitrilase (hydratase) and an amidase. Acrylate is then degraded by hydration to either lactate or P-hydroxypropionate. The nitrilase or amidase is also capable of hydrolyzing the nitrile group in a number of other nitriles (Robertson et al. 2004) including PAN (Tauber et al. 2000). 

Gasson Ml, Y Kitamura, WR McLaughlan, A Narbad, AJ Parr, ELH Parsons, J Payne, MJC Rhodes, NK Walton (1998) Metabolism of ferulic acid to vanillin. A bacterial gene of the enoyl-SCoA hydratase/ isomerase superfamily encodes an enzyme for the hydration and cleavage of a hydroxycinnamic acid SCoA thioester. J Biol Chem 273 4163-4170. 

Nitrile hydratase (NHase) catalyzes the hydration of nitriles to amides (Figure 1.11) and has been used for production of acrylamide and nicotinamide at large scale. NHases are roughly... 

This enzyme [EC 4.2.1.34], also known as mesaconase and mesaconate hydratase, catalyzes the conversion of (5 )-2-methylmalate to 2-methylfumarate and water. The enzyme will also catalyze the hydration of fumarate to (5 )-malate. 

Nitriles are interesting precursors of both amides and carboxylic acids. In vivo there are two pathways for the bioconversion of nitriles to carboxylic acids (Scheme 6.19). In the first method a nitrilase catalyzes the enantioselechve hydrolysis of a racemic or prochiral nitrile. The second pathway involves a two-enzyme cascade in which an aselective nitrile hydratase (NHase) catalyzes the hydration of the racemic nitrile to the racemic amide followed by an amidase-catalyzed enantioselechve hydrolysis to the carboxylic acid. The amidase is generally, but not always, (S)-selechve, resulting in the formahon of a 1 1 mixture of the (S)-acid... 

The lyases comprise enzyme class 4. They are enzymes cleaving C-C, C-0, C-N and other bonds by elimination, not by hydrolysis or oxidation. Lyases also catalyse addition to donble bonds. The types of reactions catalysed by lyases are decarboxylation (decarboxylase), hydration/dehydration (hydratase/dehydratase), ammonia addition/deamination (ammonia-lyase), cyanohydrin formation/cleavage (oxynitrilase),... 

As well as detoxication via reaction with GSH, the reactive 3,4-epoxide can be removed by hydration to form the dihydrodiol, a reaction that is catalyzed by epoxide hydrolase (also known as epoxide hydratase). This enzyme is induced by pretreatment of animals with the polycyclic hydrocarbon 3-methylcholanthrene, as can be seen from the increased excretion of 4-bromophenyldihydrodiol (Table 7.5). This induction of a detoxication pathway offers a partial explanation for the decreased hepatotoxicity of bromobenzene observed in such animals. A further explanation, also apparent from the urinary metabolites, is the induction of the form of cytochrome P-450 that catalyzes the formation of the 2,3-epoxide. This potentially reactive metabolite readily rearranges to 2-bromophenol, and hence there is increased excretion of 2-bromophenol in these pretreated animals (Table 7.5). 

Fumarate is hydrated to malate in a freely reversible reaction cat alyzed by fumarase (also called fumarate hydratase, see Figure 9.6). [Note- Fumarate is also produced by the urea cycle (see p. 251), in purine synthesis (see p. 293), and during catabolism of the amino acids, phenylalanine and tyrosine (see p. 261).]... 

Succinyl CoA is cleaved by succinate thiokinase (also called succinyl CoA synthetase), producing succinate and ATP (or GTP). This is an example of substrate-level phosphory lation. Succinate is oxidized to fumarate by succinate dehydrogenase, producing FADH2. The enzyme is inhibited by oxaloacetate. Fumarate is hydrated to malate by fumarase (fumarate hydratase), and malate is oxidized to oxaloacetate by malate dehy drogenase, producing NADH. 

Fumarate hydratase (fumarase), which is discussed in Chapter 13, catalyzes the reversible hydration of fumaric acid to malic acid (Eq. 13-11). It was one of the first enzymes whose pH dependence was studied intensively. A bell-shaped pH dependence... 

Another example is provided by malic acid, a chiral molecule which also contains a prochiral center (see Eq. 9-74). In this case replacement of the pro-R or pro-S hydrogen atom by another atom or group would yield a pair of diastereoisomers rather than enantiomers. Therefore, these hydrogen atoms are diastereotopic. When L-malic acid is dehydrated by fumarate hydratase (Chapter 13) the hydrogen in the pro-R position is removed but that in the pro-S position is not touched. This can be demonstrated by allowing the dehydration product, fumarate, to be hydrated to malate in 2HzO (Eq. 9-74). The malate formed contains deuterium in the pro-R position. If this malate is now isolated and placed with another portion of enzyme in H20, the deuterium is removed cleanly. The fumarate produced contains no deuterium. 

Many addition and elimination reactions, e.g., the hydration of aldehydes and ketones, and reactions catalyzed by lyases such as fumarate hydratase are strictly reversible. However, biosynthetic sequences are often nearly irreversible because of the elimination of inorganic phosphate or pyrophosphate ions. Both of these ions occur in low concentrations within cells so that the reverse reaction does not tend to take place. In decarboxylative eliminations, carbon dioxide is produced and reversal becomes unlikely because of the high stability of C02. Further irreversibility is introduced when the major product is an aromatic ring, as in the formation of phenylpyruvate. 

Again, these appear to resemble the corresponding Mo-dependent enzymes. The unique acetylene hydratase from the acetylene-utilizing Pelobacter acetylenicus catalyzes the hydration of acetylene to acetaldehyde.687... 

Acetylene hydratase [74] is unique among the molybdenum and tungsten enzymes. This enzyme catalyzes the hydration of an unsaturated organic substrate, acetylene, a reaction that is neither an oxidation nor a reduction. Although tungsten may assume different oxidation states, the non-redox nature of the sub-... 

Hydration and/or dehydration reactions are frequently catalyzed by metallopro-teins. Examples are proteins containing nickel (urease), zinc (e.g., peptidases), molybdenum (the hydratase partial reaction of formate oxidoreductase), tungsten (acetylene hydratase). An obvious difference between Ni, Zn, on the one hand, and Fe, Mo, W, on the other, is that the first are directly coordinated to the protein whereas the latter are also part of a cofactor. With reference to the Fe/S cluster in aconitase it has been suggested that cofactor coordination may provide an added flexibility to the active site, in particular to the substrate binding domain [15],... 

Hydration of the trans A2-enoyl CoA to form 3-hydroxyacyl CoA (catalyzed by enoyl CoA hydratase). 

Another numerical experiment involved a model active site of Fe-dependent nitrile-hydratase (Nhase), a non-heme Fem enzyme that catalyzes the hydration of nitriles to amides. The mechanism is as yet unknown and computational chemistry may be important to help unraveling it, provided the methods used are adequate, and the idea was that calculating the spectrum is a good check on the computational method. Of the model compound, [Fem(PyPepS)2r (see Figure 3-12) the spectrum in aqueous solution is known. 

Step 7 is the reversible hydration of fumarate to form malate, catalyzed by fumarate hydratase (which is usually called fumarase). 

Epoxide rings of certain alkene and arene compounds are hydrated enzymatically by epoxide hydrolases to form the corresponding iram-dihydrodiols (Figure 10.11). The epoxide hydrolases are a family of enzymes known to exist both in the endoplasmic reticulum and in the cytosol. In earlier studies they were named epoxide hydratase, epoxide hydrase, or epoxide hydrolase. Epoxide hydrolase, however, has been recommended by the International Union of Biochemists Nomenclature Committee and is now in general use. 

Another example in which a biocatalytic transformation has replaced a chemo-catalytic one, in a very simple reaction, is the Mitsubishi Rayon process for the production of acrylamide by hydration of acrylonitrile (Fig. 1.42). Whole cells of Rhodococcus rhodocrous, containing a nitrile hydratase, produced acrylamide in >99.9% purity at >99.9% conversion, and in high volumetric and space time yields [121]. The process (Fig. 1.42) currently accounts for more than 100000 tons annual production of acrylamide and replaced an existing process which employed a copper catalyst. A major advantage of the biocatalytic process is the high product purity, which is important for the main application of acrylamide as a specialty monomer. 

Similarly, DuPont employs a nitrile hydratase (as whole cells of P. chlororaphis B23) to convert adiponitrile to 5-cyanovaleramide, a herbicide intermediate [122]. In the Lonza nitrotinamide (vitamin B6) process [123] the final step (Fig. 1.42) involves the nitrile hydratase (whole cells of Rh. rhodocrous) catalysed hydration of 3-cyanopyridine. Here again the very high product purity is a major advantage as conventional chemical hydrolysis affords a product contaminated with nicotinic acid, which requires expensive purification to meet the specifications of this vitamin. 

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