Bacterial dehydratases

The role of the iron-sulfur clusters in many of the proteins that we have just considered is primarily one of single-electron transfer. The Fe-S cluster is a place for an electron to rest while waiting for a chance to react. There may sometimes be an associated proton pumping action. In a second group of enzymes, exemplified by aconitase (Fig. 13-4), an iron atom of a cluster functions as a Lewis acid in facilitating removal of an -OF group in an a,P dehydration of a carboxylic acid (Chapter 13). A substantial number of other bacterial dehydratases as well as an important plant dihydroxyacid dehydratase also apparently use Fe-S clusters in a catalytic fashion.317 Fumarases A and B from E. coli,317 L-serine dehydratase of a Pepto-streptococcus species,317-319 and the dihydroxyacid... 

Song J, T Xia, RA Jensen (1999) PhhB, a Pseudomonas aeruginosa homolog of mammalian pterin 4a-carbi-nolamine dehydratase/DCcoH, does not regulate expression of phenylalanine hydroxylase at the transcriptional level. J Bacterial 181 2789-2796. 

One of the first known examples of allosteric feedback inhibition was the bacterial enzyme system that catalyzes the conversion of L-threonine to L-isoleucine in five steps (Fig. 6-28). In this system, the first enzyme, threonine dehydratase, is inhibited by isoleucine, the product of the last reaction of the series. This is an example of heterotropic allosteric inhibition. Isoleucine is quite specific as an inhibitor. No other intermediate in this sequence inhibits threonine dehydratase, nor is any other enzyme in the sequence inhibited by isoleucine. Isoleucine binds not to the active site but to another specific site on the enzyme molecule, the regulatory site. This binding is noncovalent and readily reversible if the isoleucine concentration decreases, the rate of threonine dehydration increases. Thus threonine dehydratase activity responds rapidly and reversibly to fluctuations in the cellular concentration of isoleucine. 

Other enzymes in the aconitase family include isopropylmalate isomerase and homoaconitase enzymes functioning in the chain elongation pathways to leucine and lysine, both of which are pictured in Fig. 17-18.90 There are also iron-sulfur dehydratases, some of which may function by a mechanism similar to that of aconitase. Among these are the two fumarate hydratases, fumarases A and B, which are formed in place of fumarase C by cells of E. coli growing anaerobically.9192 Also related may be bacterial L-serine and L-threonine dehydratases. These function without the coenzyme pyridoxal phosphate (Chapter 14) but contain iron-sulfur centers.93-95 A lactyl-CoA... 

H. J. Blumenthal and T. Jepson, Asymmetric dehydration of galactarate by bacterial galactarate dehydratase, Biochem. Biophys. Res. Commun., 17 (1964) 282-287. 

A similar stereochemical question as in the /8-replacement reactions can be asked in the a, /8-eliminations where the group X is replaced by a hydrogen, i.e., is the proton added at C-/8 of the PLP-aminoacrylate on the same face from which X departed or on the opposite face This question has been answered for a number of enzymes which generate either a-ketobutyrate or pyruvate as the keto acid product. Crout and coworkers [119,120] determined the steric course of proton addition in the a,/8-elimination of L-threonine by biosynthetic L-threonine dehydratase and of D-threonine by an inducible D-threonine dehydratase, both in Serratia marcescens. Either substrate, deuterated at C-3, was converted in vivo into isoleucine, which was compared by proton NMR to a sample prepared from (3S)-2-amino[3-2H]butyric acid. With both enzymes the hydroxyl group at C-3 was replaced by a proton in a retention mode. Although this has not been established with certainty, it is likely that both enzymes, like other bacterial threonine dehydratases [121], contain PLP as cofactor. Sheep liver L-threonine dehydratase, on the other hand, is not a PLP enzyme but contains an a-ketobutyrate moiety at the active site [122], It replaces the hydroxyl group of L-threonine with H in a retention mode, but that of L-allothreonine in an inversion mode [123]. Snell and coworkers [124] established that the replacement of OH by H in the a, /8-elimination of D-threonine catalyzed by the PLP-containing D-serine dehydratase from E. coli also proceeds in a retention mode. They... 

Trapping of the aminoacrylate intermediate in the reactions catalyzed by cystathionine-y-synthase and y-cystathionase produced the same diastereomer of KEDB which was different from the one formed with bacterial L-threonine dehydratase. Unfortunately, this experiment has apparently not been done with threonine synthetase. 

Over the past decade, several strains of yeast [43, 44] and E. coli [45, 46] have been engineered that lack chorismate mutase. A typical bacterial selection system is depicted schematically in Fig. 3.5. It is based on E. coli strain KA12 [45], which has deletions of the chromosomal genes for both bifunctional chorismate mutases (chorismate mutase-prephenate dehydrogenase and chorismate mutase-prephenate dehydratase). Monofunctional versions of prephenate dehydratase [47] and prephenate dehydrogenase [48] from other organisms are supplied by the plasmid pKIMP-UAUC, leaving the cells deficient only in chorismate mutase activity [45]. 

Leesong M, Henderson BS, Gillig JR, Schwab JM, Smith JL. Structure of a dehydratase-isomerase from the bacterial pathway for biosynthesis of unsaturated fatty acids two catalytic activities in one active site. Structure 1996 4 253-264. 

On the other hand, the pathways to both aromatic amino acids, Phe (1)/Tyr (2), in vascular plants are only beginning to be clarified, and could not readily be deduced from bacterial/fungal sequence/comparisons, for example, in terms of substrate(s). Thus the key to fully understanding the pathways to these two aromatic amino acids in plants was to identify the enzymes, as well as to ultimately establish their substrate specificities/ feedback properties that is, of the actual dehydratases, dehydrogenases, and aromatic aminotransferases involved, including how transcriptional regulation is attained. 

Bacterial Prephenate Dehydratases and Plant Arogenate Dehydratases ... 

Since the PKS (polyketide synthase) gene cluster for actinorhodin (act), an antibiotic produced by Streptomyces coelicolor[ 109], was cloned, more than 20 different gene clusters encoding polyketide biosynthetic enzymes have been isolated from various organisms, mostly actinomycetes, and characterized [98, 100]. Bacterial PKSs are classified into two broad types based on gene organization and biosynthetic mechanisms [98, 100, 102]. In modular PKSs (or type I), discrete multifunctional enzymes control the sequential addition of thioester units and their subsequent modification to produce macrocyclic compounds (or complex polyketides). Type I PKSs are exemplified by 6-deoxyerythronolide B synthase (DEBS), which catalyzes the formation of the macrolactone portion of erythromycin A, an antibiotic produced by Saccharopolyspora erythraea. There are 7 different active-site domains in DEBS, but a given module contains only 3 to 6 active sites. Three domains, acyl carrier protein (ACP), acyltransferase (AT), and P-ketoacyl-ACP synthase (KS), constitute a minimum module. Some modules contain additional domains for reduction of p-carbons, e.g., P-ketoacyl-ACP reductase (KR), dehydratase (DH), and enoyl reductase (ER). The thioesterase-cyclase (TE) protein is present only at the end of module 6. 

Evidence for the presence of the enzymes of the histidine pathway in plants appears to be limited to the work of Winter et al. (1971a) who demonstrated the presence of ATP-phosphoribosyltransferase, the first enzyme of the pathway, imidazole glycerolphosphate dehydratase and histidinol phosphatase in extracts from the shoots of barley, oats, and peas, and to the unpublished observations of Davies (see Davies, 1971) on the presence of histidinol dehydrogenase in rose tissue culture cells. The specific activity of ATP-phosphoribosyltransferase was greatest in peas and oats and least in barley. The enzymes from oats and barley were thermolabile losing activity after 30 min at 37°C. The specific activities of imidazole glycerolphosphate dehydratase were very low but it was possible to purify the enzyme to some extent. The values for imidazole glycerolphosphate for the barley enzyme was 0.6 mM which compares with values for jthe yeast and bacterial enzymes of 0.3 and 0.4 mM, respectively. Histidinolphosphatase was purified 20-fold but the authors considered that two phosphatases were still present. 

Enzymes involved as catalysts in each of the steps from 3-dehydro-quinate (10) to chorismate (14) in the conunon pathway have all been subsequently isolated and characterised from bacterial mutants. Methods of assay for each form of activity have been described . Mitsuhashi and Davis first isolated 3-dehydroquinate dehydratase (E.C. 4.1.2.10) the enzyme which is responsible for the dehydration step (10 11). With a partially purified extract they showed that the... 

Certain bacterial strains prodnce bitterness in wine—a fact known since the time of Pasteur. Lactic acid bacteria make use of a glycerol dehydratase to transfonn glycerol into j0-hydroxy-propionaldehyde (Figure 5.8). This molecule is the precursor of acrolein, which is formed in wine by heating, or slowly during aging. The combination of wine tannins and acrolein, or its precursor, gives a bitter taste. 

---