PLP-dependent lyases,

Extensive comparative studies of several PLP-dependent lyases Involved in the synthesis and transformations of cysteine, serine and their natural or synthetic analogues have been conducted during the last decennium by us and a group of associates (see Refs. " D-lif and Table 1). Such lyases may have... 

Fig, 1. Scheme of the mechanisms of o<,/-elimination and -replacement reactions catalysed by PLP-dependent lyases (after L.Davis it D.Metzler 9),... 

Table 1. PLP-Dependent Lyases Studied MECHANISMS OF PLP-DEPENDENT B-REPLACEMENT REACTIONS...

However, the situation is apparently, different for one subgroup among the PLP-dependent lyases extensively studied in recent.years in our laboratory on comparative lines,cf. surveys When highly purified, lyases belonging to this subtype, namely, cysteine lyase, serine sulfhydrase, cystathionine E-synthase, and E-cyanoalanine synthase (enzy-mes 1 - it Table 1 2 and obviously are species-specific... 

Table 2. Action of PLP-dependent lyases in presence of P -cl -N-ethylmaleimide (NEM)...

We applxed this test to B-speclflc PLP-dependent lyases which. In our opinion, might not require this step As already reported, lyases J. - it are refractory to Inhibition by L-cycloserlne (cSer) Slight affinity to the enzymes could be demonstrated at high cSer concentrations ( 0 1 M) filtrates of denatured cSer-enzyme mixtures contained coenzyme-ln-hlbltor Imlnes (reducible with to secondary amines) ... 

Monooxygenase, Isopenicillin N synthase, Glutathione peroxidase, Methylmalonyl-CoA mutase, PLP-dependent b-lyase... 

The present chapter reviews applications in biocatalysis of the ONIOM method. The focus is on studies performed in our research group, in most cases using the two-layer ONIOM(QM MM) approach as implemented in Gaussian [23], The studied systems include methane monooxygenase (MMO), ribonucleotide reductase (RNR) [24, 25], isopenicillin N synthase (IPNS) [26], mammalian Glutathione peroxidase (GPx) [27,28], Bi2-dependent methylmalonyl-CoA mutase [29] and PLP-dependent P-lyase [30], These systems will be described in more detail in the following sections. ONIOM applications to enzymatic systems performed by other research groups will be only briefly described. 

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). 

Cysteine is formed in plants and in bacteria from sulfide and serine after the latter has been acetylated by transfer of an acetyl group from acetyl-CoA (Fig. 24-25, step f). This standard PLP-dependent (3 replacement (Chapter 14) is catalyzed by cysteine synthase (O-acetylserine sulfhydrase).446 447 A similar enzyme is used by some cells to introduce sulfide ion directly into homocysteine, via either O-succinyl homoserine or O-acetyl homoserine (Fig. 24-13). In E. coli cysteine can be converted to methionine, as outlined in Eq. lb-22 and as indicated on the right side of Fig. 24-13 by the green arrows. In animals the converse process, the conversion of methionine to cysteine (gray arrows in Fig. 24-13, also Fig. 24-16), is important. Animals are unable to incorporate sulfide directly into cysteine, and this amino acid must be either provided in the diet or formed from dietary methionine. The latter process is limited, and cysteine is an essential dietary constituent for infants. The formation of cysteine from methionine occurs via the same transsulfuration pathway as in methionine synthesis in autotrophic organisms. However, the latter use cystathionine y-synthase and P-lyase while cysteine synthesis in animals uses cystathionine P-synthase and y-lyase. 

L-Serine is converted to pyruvate + NH3 by serine dehydratase (deaminase) in a PLP-dependent reaction. However, using the same coenzyme selenocysteine is converted by selenocysteine lyase into L-alanine + elemental selenium Se°. l-Cysteine may be converted by PLP-dependent enzymes into wither H2S or into S° for transfer into metal clusters. Compare the chemical mecha-... 

Tryptophanase (L-tryptophan indole-lyase (deaminating) EC 4.1.99.1) belongs to the family of the pyridoxal 5 -phosphate (PLP)-dependent enzymes. It serves in vivo to degrade L-tryptophan, is induced by L-tryptophan, and found in various bacteria, particularly in enteric species. Tryptophanase catalyzes a,/3-elimination1 and /3-replacement reactions on interaction with L-tryptophan and various other /3-substituted amino acids2 ... 

The approaches that have been described in some detail for tryptophan synthase may be applied to other PLP-dependent enzymes. Tryptophan indole lyase, or tryptophanase, catalyzes the PLP-dependent P-elimination of indole from tryptophan to yield indole, pyruvate, and NH4+ (Equation 4). 

PLP-dependent desulfhydrases necessarily show very similar mechanisms, but often come from independent evolutionary lineages. For example, although most bacterial L-cysteine desulfhydrases are fold-type I enzymes belonging to the same evolutionary branch as cystathionine f3- and 7-lyases, L-cysteine desulfhydrase from Fusobacterium nucleatum is a member of the fold-type II group and its closest sequence homologue is a cysteine synthase. D-cysteine desulfhydrase from E. coli is also a fold-type II enzyme not strictly related to other desulfhydrases but resembling instead an ACC deaminase. ... 

Several PLP-dependent enzymes catalyze elimination and replacement reactions at the y-carbon of substrates, an unusual process which provides novel routes for mechanism-based inactivation. An example of this class of enzymes is cystathionine y-synthase [0-succinylhomoserine (thiol)-lyase], which converts (7-succinyl-L-homoserine and L-cysteine to cystathionine and succinate as part of the bacterial methionine biosynthetic pathway (Walsh, 1979, p. 823). Formation of a PLP-stabilized o-carbanion intermediate activates the )8-hydrogen for abstraction, yielding j8-carbanion equivalents and allowing elimination of the y-substituent. The resulting j8,y-unsaturated intermediate serves as an electrophilic acceptor for the replacement nucleophile. Suitable manipulation of the j8-carbanion intermediate allows strategies for the design of inactivators which do not affect enzymes which abstract only the a-hydrogen. 

Autotrophic organisms synthesize methionine from asparfafe as shovm in the lower right side of Fig. 24-13. This involves fransfer of a sulfur atom from cysfeine info homocysfeine, using the carbon skeleton of homoserine, the intermediate cystathionine, and two PLP-dependent enzymes, cystathionine y-synthase and cystathionine p-lyase. This transsulfuration sequence (Fig. 24-13, Eq. 14-33) is essentially irreversible because of the cleavage to pyruvate and NH4+ by the P-lyase. Nevertheless, this transsulfuration pathway operates in reverse in the animal body, which uses two different PLP enzymes, cystathionine P s3mthase (which also contains a bound heme) and cystathionine y-lyase (Figs. 24-13,24-16, steps h and i), in a pathway that metabolizes excess methionine. 

PLP dependent enzymes belong to five enzyme classes transferases, hydrolases, lyases, isomerases and oxidoreductases. Nearly half of PLP-dependent enzymes are involved in important steps of amino acid metabolism (decarboxylation, a,p-elimination reactions, racemization and transamination). During the reaction of amino acids (and other amino compounds), the... 

The term vitamin Bg refers to a group of naturally occurring pyridine derivatives represented by pyridoxine (pyridoxol, PN), pyridoxal (PL), and pyridoxamine (PM), and their phosphorylated derivatives. They are collectively referred to as vitamin Bg vitamers. The natural free forms of the vitamers could be converted to the key coenzymatic form, pyridoxal phosphate (PLP), by the action of two enzymes, a kinase and an oxidase. There are more than 140 PLP-dependent enzymatic reactions, and they are distributed in all organisms. These enzymes comprise diverse groups such as the oxidoreductases, transferases, hydrolases, lyases, and isomerases. About... 

A variety of amino acids with electrophilic substituents (HO, RS, indolyl, etc.) in positions B or y are of general biological significance as protein constituents and metabolic intermediates, or in more restricted roles as detoxication products or secondary nitrogen reserves (in plants) In the biosynthesis and metabolism of such amino acids essential steps are catalysed by a group of widespread pyridoxal-P (PLP) dependent enzymes, classified as lyases (EC class kt see Ref.o), which effect, more or less selectively, elimination and/or replacement reactions of the X-substituent, according to equations I - IV ... 

Reactions at the j8-position (for example, in threonine dehydatase) or the y-position (in methionine-y-lyase) also proceed by means of formation of an aldimine intermediate with the Q -carbon of an a-amino acid. Such a survey of PLP-dependent enzymes illustrates the important point that one cofactor can be used for different kinds of transformations. The reactions described all go through a common aldimine intermediate, with the ultimate course of the reaction being controlled by the appropriate substrate specificity and positioning of amino acid side chains. This flexibility allows nature to expand its chemical repertoire with a relatively small set of cofactors. 

---