E. coli enzymes

The E. coli enzyme accepts substitution on either cosubstrate propanal, acetone or 1-fluoro-2-propanone can replace the donor and a variety of aldehydes can replace the acceptor moiety 3. Shortcomings are the relatively low conversion rates obtained for any substrate analog and the as yet unidentified level of relative stereocontrol induced upon substitution at the nucleophilic carbon. 

Interestingly, the E. coli enzyme s relaxed acceptor specificity allows for substitution of both cosubstrates, albeit at strongly reduced (<1% of v, catalytic rates. Propanal, acetone, or fluoroacetone can replace ethanal as the donor in the synthesis of variously substituted 3-hydroxyketones such as (112) or (113) (Figure 10.41)... 

The dihydrofolate reductase enzyme (DHFR) is involved in one-carbon metabolism and is required for the survival of prokaryotic and eukaryotic cells. The enzyme catalyzes the reduction of dihydrofolate to tetrahydrofolate, which is required for the biosynthesis of serine, methionine, purines, and thymidylate. The mouse dihydrofolate reductase (mDHFR) is a small (21 kD), monomeric enzyme that is highly homologous to the E. coli enzyme (29% identify) (Pelletier et al., 1998). The three-dimensional structure of DHFR indicates that it is comprised of three structural fragments F[l], F[2] andF[3] (Gegg etal., 1997). 

One last class of mononuclear non-haem iron enzyme that we have not yet considered, consists of the microbial superoxide dismutases with Fe(III) at their active site. The crystal structure of the E. coli enzyme shows a coordination geometry reminiscent of protocatechuate 3,4-dioxygenase, with four endogenous protein ligands, three His and one Asp residue, and one bound water molecule (Carlioz et ah, 1988). 

Another way of perturbing the commitments is solvent deuteration. Change of the carbon kinetic isotope effect in the case of E. coli enzyme indicates that the proton transfer precedes the decarboxylation step ... 

Crystal structure determinations of MnSODs from organisms ranging from E. coli to humans have been reported. Structural determinations of note include those by Jameson et al. on the E. coli enzyme and mutant forms of this enzyme with atomic resolution,a cambialistic superoxide dismutase from Porphyromonas gingivalis, and mutant forms of the human enzyme the Q143N, and Q143A mutants.The coordination sphere of the... 

C-methyl-D-erythritol 2,4-cyclodiphosphate synthase catalyses the conversion of 4-diphospho-cytidyl-2-C-methyl-D-erythritol 2-phosphate to 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MECDP) (Equation (7)). This reaction is part of the isoprenoid biosynthesis pathway in many plants and bacteria. The structure of the E. coli enzyme bound to Mn, cytosine monophosphate, and 2-C-methyl-D-erythritol 2,4-cyclodiphosphate has been determined. The enzyme in the crystal and probably in solution is trimeric, three monomers are packed in a circular assembly with three-fold symmetry. The active site is at the interface of two adjacent monomers all the ligands bound to the Mn + come from one monomer and a MECDP molecule. The structure of this active site is shown in Figure 29 ... 

L-Fucose isomerase catalyzes the interconversion of L-fucose to L-fuculose and o-arabinose to D-ribulose. It has neither sequence nor structural similarity with the other aldose-ketose isomerases. A crystal structure of the E. coli enzyme with an L-fucitol bound in the active site shows that the active site is located in a 20 A deep pocket, at the bottom of which is a single Mn ion. Mn is bound to Oi and O2 of L-fucitol the side chains of a monodentate Glu-337, a bidentate Asp-361 (with long bonds to both oxygens), His-528 and a water molecule. ... 

This enzyme [EC 4.1.1.11] catalyzes the conversion of aspartate to j8-alanine and carbon dioxide. Pyruvate is a cofactor for the E. coli enzyme. 

The exchange catalyzed by the E. coli enzyme with glutamate occurs in the absence of ammonia and is partially... 

RNR catalyzes the conversion of ribonucleotides to deoxyribonucleo-tides. Because of this central position in generating precursors for DNA synthesis, the enzyme has been the target for pharmacological modification. The reaction has been proposed to proceed through a radical mechanism involving the cyclic transfer of a preexisting radical in the enzyme to the substrate and back (Stubbe, 1989). Several classes of RNR have been identified on the basis of the type of radical and associated metallocenter. For the purposes of this chapter, only the E. coli enzyme, which has a tyrosyl radical and a binuclear iron cluster, is considered. 

Fig. 2.2.2.1 Comparison of schematic substrate-binding site models for a) E. coli enzymes DXS [23a] and b) transketolase A [6bj. c) The cofactor ThDP with bound intermediates (hydroxyethyl HE-ThDP dihydroxyethyl DHE-ThDP). Putative interactions between amino acid residues and bound acceptor substrates are indicated by dotted lines.

E. coli enzyme. But, studies on the chicken liver enzyme may not be relevant to the human enzyme. Indeed, crystal studies of Oefner et al. (1988) on complexes of human DHFR with folate, methotrexate and trimethoprim itself do not support the explanation given by Matthews, and so we are still not sure of the basis for the species selectivity exhibited by trimethoprim. 

A wild-type DERA with less than 30% sequence identity to the E. coli enzyme led to a significant improvement over the E. coli wild-type DERA-based process originally described by Wong [15], The application of rational mutagenesis, based on the available crystal structure of the E. coli enzyme, expanded the range of suitable acceptor substrates for DERA to azido-substituted aldehydes, further facilitating access to, for example, the atorvastatin side chain [16]. 

FIGURE 22-17 Biosynthesis of tryptophan from chorismate in bacteria and plants. In E. coli, enzymes catalyzing steps (T) and are subunits of a single complex. 

Therefore, to achieve high conversion of the substrate a tenfold excess of pyruvate is usually needed. The enzymes from Clostridium perfringens and Escherichia coli are commercially available from Toyobo the E. coli enzyme has been cloned and overexpressed, which has considerably reduced its cost [22,23], Sodium borohydride inactivates the enzyme in the presence of either sialic acid or pyruvate, indicating that the enzyme belongs to the Schiff-base-forming class 1 aldolase. This aldolase was supposed to be a... 

Other bacterial mutants, such as a mutant Escherichia coli enzyme tolerant to glyphosate, have been described (47). The E. coli mutant had a single amino acid change from the wild type, resulting in substitution of glycine 96 with alanine. An identical mutation was reported in glyphosate-tolerant Klebsiella pneumoniae (48). The nature of changes in the kinetic constants of the K pneumoniae enzyme is similar to that of the E. coli enzyme. 

E. coli enzyme,107 108 causing isocitrate to build up in the citric acid cycle (Fig. 10-6) and to be diverted into the glyoxylate pathway, which is depicted in Fig. 17-16. In this instance, it appears likely that the negative charge of the added phospho group causes electrostatic repulsion of the substrate isocitrate. In agreement with this concept, mutation of Ser 113 of this enzyme to Asp (mutant S113 D) also inactivates the enzyme.109... 

In fact, kinetic studies of the GTP-dependent avian mitochondrial enzyme indicate two metal-binding sites, one on the polyphosphate group of the bound GTP and one on carboxylate side chains of the protein.252 255 The three-dimensional structure of the ATP-dependent E. coli enzyme reveals a nucleotide binding site similar to the ATP site of adenylate kinase (Fig. 12-30).256 A definite binding site for C02 is also present in the enzyme.257... 

The much studied E. coli enzyme is composed of a 156-residue biotin carboxyl carrier protein,38 a 449-residue biotin carboxylase, whose three-dimensional structure in known 39/393 and a carboxyltransferase subunit consisting of 304 (a)- and 319 (P)- residue chains. These all associate as a dimer of the three subunits (eight peptide chains).40-42... 

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