The N-terminal domain is structurally very similar to class I aminoacyl-tRNA synthetases and is thus a member of the cytidylyltransferase superfamily. This relationship has been used to suggest the location of the ATP and pantoate binding sites and the nature of hinge bending that leads to the ternary enzyme-pantoate-ATP complex.

Phosphopantetheine adenylyltransferase (PPAT) catalyzes the penultimate step in coenzyme A (CoA) biosynthesis: the reversible adenylation of 4-phosphopantetheine yielding 3-dephospho-CoA and pyrophosphate. Wild-type PPAT from Escherichia coli was purified to homogeneity. N-terminal sequence analysis revealed that the enzyme is encoded by a gene designated kdtB, purported to encode a protein involved in lipopolysaccharide core biosynthesis. The gene, here renamed coaD, is found in a wide range of microorganisms, indicating that it plays a key role in the synthesis of 3-dephospho-CoA. Overexpression of coaD yielded highly purified recombinant PPAT, which is a homohexamer of 108 kDa. Not less than 50% of the purified enzyme was found to be associated with CoA, and a method was developed for its removal. A steady state kinetic analysis of the reverse reaction revealed that the mechanism of PPAT involves a ternary complex of enzyme and substrates. Since purified PPAT lacks dephospho-CoA kinase activity, the two final steps of CoA biosynthesis in E. coli must be catalyzed by separate enzymes.Coenzyme A (CoA) 1 is an essential cofactor in numerous biosynthetic, degradative, and energy-yielding metabolic pathways and is involved in the control of several key reactions in intermediary metabolism (1). CoA also donates the 4Ј-phosphopantetheinyl cofactor to the acyl carrier protein of the fatty acid synthase complex (2).The synthesis of CoA occurs in five steps which, utilize pantothenate (vitamin B 5 ), cysteine, and ATP (for review, see Ref.3). In all systems studied, the rate of CoA biosynthesis appears to be regulated by feedback inhibition of the first enzyme of the pathway, pantothenate kinase (4 -7). In vitro studies of pantothenate kinase from Escherichia coli showed that (a) CoA and, to a lesser extent, its acyl thioesters are competitive inhibitors with respect to ATP and (b) the K i values are within the physiological range of intracellular CoA concentrations (6). Studies of the intermediates in CoA biosynthesis have shown that both pantothenate and 4Ј-phosphopantetheine can accumulate in the cell (8). Hence, in addition to control of CoA synthesis on the level of pantothenate kinase, further modulation of flux through the pathway could occur at phosphopantetheine adenylyltransferase (PPAT), which catalyzes the penultimate step in the pathway (Fig. 1), the reversible adenylation of 4Ј-phosphopantetheine to form 3Ј-dephospho-CoA (dPCoA) and pyrophosphate (PP i ). Regulation at this step may control the reutilization of 4Ј-phosphopantetheine arising either from the turnover of the 4Ј-phosphopantetheinyl cofactor of the acyl carrier protein (8) or the cleavage of CoA by a phosphodiesterase (9).Despite the above arguments for a role in the regulation of CoA biosynthesis, PPAT has not been the subject of a detailed study. Enzymes with PPAT activity have been purified from a number of different organisms. In mammals PPAT has been shown to be part of a complex that also includes dPCoA kinase, the effector of the fina...

The role of conserved Asp-199 in chloramphenicol acetyltransferase (CAT) has been investigated by site-directed mutagenesis. Substitution of Asp-199 by alanine results in a thermolabile mutant enzyme (Ala-199 CAT) with reduced kcat(13-fold) but similar Km values to wild type CAT. Replacement by asparagine gives rise to a thermostable mutant enzyme (Asn-199 CAT) with much reduced kcat(1500-fold). Furthermore, Asn-199 CAT shows anomalous inactivation kinetics with the affinity reagent 3-(bromo-acetyl)chloramphenicol. These results favor a structural role for Asp-199 rather than a catalytic one, in keeping with crystallographic evidence for involvement of Asp-199 in a tight salt bridge with Arg-18. Replacement of Arg-18 by valine results in a mutant enzyme (Val-18 CAT) with similar properties to Ala-199 CAT. The catalytic imidazole of His-19 appears to be conformationally constrained by hydrogen bonding between N1-H and the carbonyl oxygen of the same residue and by ring stacking with Tyr-25.

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The imidazole N epsilon 2 of His-195 plays an essential part in the proposed general base mechanism of chloramphenicol acetyltransferase (CAT), hydrogen bonding to and a abstracting a proton from the primary hydroxyl group of chloramphenicol. Replacement of His-195 by alanine or glutamine results in apparent decreases in kcat of (9 x 10(5)- and (3 x 10(5))-fold, respectively, whereas Km values for both substrates (chloramphenicol and acetyl-CoA) are similar to those of wild-type CAT. The structure of Gln-195 CAT has been solved at 2.5-A resolution and is largely isosteric with that of wild-type CAT. Substitution of His-195 by glutamate resulted in a (5 x 10(4))-fold decrease in kcat together with a 3-fold increase in the Km for chloramphenicol. Direct determination of binding constants for both substrates demonstrated that these substitutions result in only small decreases in the affinity of CAT for acetyl-CoA (Kd values increased 2- to 3-fold), whereas chloramphenicol Kd values are elevated 26-, 20-, and 53-fold for Ala-195 CAT, Gln-195 CAT and Glu-195 CAT, respectively. The pH dependence of kcat/Km yields apparent pKa values of 6.5 and 6.7 for Ala-195 CAT and Gln-195 CAT, respectively, which are very similar to that (6.6) determined for the ionization of His-195 in wild-type CAT. In contrast, the pH dependence of kcat/Km for Glu-195 CAT (pKa = 8.3) is very different from that of wild-type CAT.(ABSTRACT TRUNCATED AT 250 WORDS)