Pyridoxal-dependent decarboxylases

Drosophila DDC belongs to a family of pyridoxal-dependent decarboxylases that extends from prokaryotes to eukaryotic plants and animals. The members of this family show significant sequence similarity over much of their length, even though the individual proteins have quite different substrate specificities, including the amino acids tyrosine, tryptophan, phenylalanine, histidine, and glutamate, and the amino acid derivatives... 

Jackson, F. R. (1990). Prokaryotic and eukaryotic pyridoxal-dependent decarboxylases are homologous. J. Mol. Evol. 31 325-329. 

Decarboxylation of an amino acid is an important reaction, catalyzed by a pyridoxal-dependent decarboxylase, that affords an amine as product (Scheme 2.6). It is very attractive to learn how to mimic this process to generate various amines from a-amino acids. Unfortunately, our previous studies established that treatment of a-alkyl amino acids with pyridoxal afforded only ketone and pyridoxamine as products, by a transamination-dependent oxidative decarboxylation process (pathway b in Scheme 2.5) [41]. Consequently, non-oxidative decarboxylation, using pyridoxals to generate amines, remains elusive. 

There have been k12/k13 KIEs measured on C02 release (1.033) by this enzyme which indicate that the ratio kjk5 is not very different from unity, i.e. that transimination and decarboxylation are both partially rate-limiting147. Based on a comparison of a variety of KIEs, as well as steady-state kinetic parameters Vmax and VmaJKM for the pyruvyl-dependent and pyridoxal-dependent decarboxylases, no obvious reasons could be found why nature would preferentially select one pathway over the other. 

Ornithine decarboxylase is a pyridoxal dependent enzyme. In its catalytic cycle, it normally converts ornithine (7) to putrisine by decarboxylation. If it starts the process with eflornithine instead, the key imine anion (11) produced by decarboxylation can either alkylate the enzyme directly by displacement of either fluorine atom or it can eject a fluorine atom to produce viny-logue 12 which can alkylate the enzyme by conjugate addidon. In either case, 13 results in which the active site of the enzyme is alkylated and unable to continue processing substrate. The net result is a downturn in the synthesis of cellular polyamine production and a decrease in growth rate. Eflornithine is described as being useful in the treatment of benign prostatic hyperplasia, as an antiprotozoal or an antineoplastic substance [3,4]. 

Scheme 18.45 Postulated inhibition mechanism of pyridoxal phosphate-dependent decarboxylases by a-allenic a-amino acids.

Figure 1. Pyridoxal Phosphate Dependent Decarboxylase Mechanism...

S.2.3.3 Treatment of Trypanosomiasis The difluoromethylornithine (DFMO), eflomithine is a mechanism-based inhibitor of ornithine decarboxylase— a pyridoxal-dependent key enzyme of the polyamine s biosynthesis from ornithine. Fluorine atoms are essential for the inhibition process (cf. Chapter 7). Eflornithine was first clinically developed for cancer, but its development has been abandoned for this indication. The activity of eflornithine on trypanosomes was then discovered. Now, despite its very low bioavailability, eflornithine is the best therapy for sleeeping sickness (trypanosomiasis)—in particular, at the cerebral stage—due to Trypanosoma brucei gambiense parasite. Eflornithine is registered with orphan drug status and is distributed by the WHO. 

One of the earliest published attempts to create antibodies with catalytic activity had as its goal the generation of a transaminase. Raso and Stollar prepared V-(5-phosphopyridoxyl)-3 -amino-L-tyrosine 154 as a mimic of the Schiff s base intermediate that is formed during the pyridoxal-dependent transamination of tyrosine and showed that it was a site-directed inhibitor of the enzymes tyrosine transaminase and tyrosine decarboxylase.132 Partially purified polyclonal antibodies, elicited against y-globulin conjugates of the hapten, recognized both the... 

Table 9.2 Amines Formed by Pyridoxal Phosphate-Dependent Decarboxylases...

All terpenoid indole alkaloids are derived from tryptophan and the iridoid terpene secologanin (Fig. 2b). Tryptophan decarboxylase, a pyridoxal-dependent enzyme, converts tryptophan to tryptamine (62, 63). The enzyme strictosidine synthase catalyzes a stereoselective Pictet-Spengler condensation between tryptamine and secologanin to yield strictosidine. Strictosidine synthase (64) has been cloned from the plants C. roseus (65), Rauwolfla serpentine (66), and, recently, Ophiorrhiza pumila (67). A crystal structure of strictosidine synthase from R. serpentina has been reported (68, 69), and the substrate specificity of the enzyme can be modulated (70). 

According to O Leary the A /A KIE (for loss of COj) varies between 1.01 and 1.03 for a variety of pyridoxal phosphate-dependent decarboxylases . Assuming (based on models) that the intrinsic KIE is ca 1,05 for the decarboxylation step these numbers suggest that decarboxylation is not totally rate-determining, rather Schiff-base interchange (transimination) and C—C scission are both participating in rate-limitation ki a s) see Scheme 13. 

When purified, the DO PA decarboxylase of rat liver has an absorption spectrum similar to that of other pyridoxal-dependent enzymes. In this case, the co-enzyme seems to be very tightly bound to the apo-enzyme, but addition of an excess of pyridoxal phosphate still causes an increase in the enzyme activityii . It was therefore suggested that pyridoxal phosphate is a prosthetic group of this enzyme, and that when present in excess it acts as a co-enzyme. The 5-HTP decarboxylase of rat kidney was found to be potentiated by pyridoxal phosphate, but the effect was shown only when the tissue had been repeatedly frozen and thawed. These observations provide some evidence that pyridoxal phosphate is the co-enzyme for non-specific histidine decarboxylase. 

Pyridoxal phosphate is a co-enzyme for numerous enzymes, notably amino acid decarboxylases, amino acid transaminases, histaminase and probably diamine oxidase Ais.iw. As most of the evidence on which the mechanism of action of pyridoxal-dependent enzymes is based has been obtained from studies of the non-enzymic interaction of pyridoxal with amino acids, these non-enzymic reactions will be considered first in some detail. 

Carbonyl reagents, including cyanide, hydroxylamine, semicarbazide, hydrazine and substituted hydrazines inhibit non-specific histidine decarboxylase by combining with the co-enzyme pyridoxal phosphate. Such compounds, of course, inhibit other pyridoxal-dependent enzymes. A list of these and other compounds which inhibit non-specific histidine decarboxylase has been compiled by Schayer . 

The histidine decarboxylase activity of tissues can be raised or lowered by changing the hormonal state of the animal or by subjecting it to certain stressful stimuli. Administration of thyroid hormones to rats produces a marked increase in the specific histidine decarboxylase activity of the glandular mucosa of the stomach " , while the activity of the nonspecific enzyme in the liver is lowered . Studies of the action of thyroid hormones on other pyridoxal-dependent enzymes in rat liver suggest that, in this organ at least, these changes arise from corresponding alterations in both pyridoxal phosphate and apo-enzyme synthesis . 

Decarboxylation is one of the most common processes in natural metabolism. All decarboxylases [EC 4.1.1.-] cleave a substrate carboxylic group with or without the requirement of an enzymatic cofactor. There are three known decarboxylase types (i) thiamine diphosphate (ThDP)-dependent decarboxylases, (ii) pyridoxal phosphate (PLP)-dependent decarboxylases, and (iii) cofactor-independent decarboxylases (Figure 3.1) [1-4]. Cofactor-independent decarboxylases are specific for activated substrates. 

Histamine is synthesised by decarboxylation of histidine, its amino-acid precursor, by the specific enzyme histidine decarboxylase, which like glutaminic acid decarboxylase requires pyridoxal phosphate as co-factor. Histidine is a poor substrate for the L-amino-acid decarboxylase responsible for DA and NA synthesis. The synthesis of histamine in the brain can be increased by the administration of histidine, so its decarboxylase is presumably not saturated normally, but it can be inhibited by a fluoromethylhistidine. No high-affinity neuronal uptake has been demonstrated for histamine although after initial metabolism by histamine A-methyl transferase to 3-methylhistamine, it is deaminated by intraneuronal MAOb to 3-methylimidazole acetic acid (Fig. 13.4). A Ca +-dependent KCl-induced release of histamine has been demonstrated by microdialysis in the rat hypothalamus (Russell et al. 1990) but its overflow in some areas, such as the striatum, is neither increased by KCl nor reduced by tetradotoxin and probably comes from mast cells. 

Kamath AV, GL Vaaler, EE Snell (1991) Pyridoxal phosphate-dependent histidine decarboxylases. Cloning, sequencing, and expression of genes from Klebsiella planticola and Enterobacter aerogenes and properties of the overexpressed enzymes. J Biol Chem 266 9432-9437. 

The other enzyme involved in the synthesis of 5-HT, aromatic L-amino acid decarboxylase (AADC) (EC 4.1.1.28), is a soluble pyridoxal-5 -phosphate-dependent enzyme, which converts 5-HTP to 5-HT (Fig. 13-5). It has been demonstrated that administration of pyridoxine increases the rate of synthesis of 5-HT in monkey brain, as revealed using position emission tomography (this technique is discussed in Ch. 58). This presumably reflects a regulatory effect of pyridoxine on AADC activity and raises the interesting issue of the use of pyridoxine supplementation in situations associated with 5-HT deficiency. 

Histamine is synthesized by decarboxylation of histidine by L-histidine decarboxylase (HDC), which is dependent on the cofactor pyridoxal-5 -phosphate [21]. Mast cells and basophils are the major source of granule-stored histamine, where it is closely associated with the anionic proteoglycans and chondroitin-4-sulfate. Histamine is released when these cells degranulate in response to various immunologic and non-immunologic stimuli. In addition, several myeloid and lymphoid cell types (DCs and T cells), which do not... 

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