The biosynthesis of coenzyme A in bacteria

VITAMINS AND HORMONES, VOL. 61

The Biosynthesis of Coenzyme A in Bacteria

TADHG P. BEGLEY, CYNTHIA KINSLAND, AND CRICK STRAUSS

Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York

I. Introduction H. The Coenzyme A Biosynthetic Pathway in Bacteria

A. Aspartate 1-Decarboxylase B. Ketopantoate Hydroxymethyltransferase C. Ketopantoate Reductase D. Pantothenate Synthase E. Pantothenate Kinase F. 4'-Phosphopantothenoylcysteine Synthase G. 4'-Phosphopautothenoylcysteine Decarboxylase H. 4'-Phosphopantetheine Adenylyltransferase I. Dephospho-Coenzyme A Kinase J. 4'-Phosphopantetheinyltransferase

III. Regulation of Coenzyme A Biosynthesis in Escherichia coli IV. Coenzyme A Biosynthesis in Saccharomyces cerevisiae V. Coenzyme A in Human Nutrition

VI. Summary and Conclusions References

Coenzyme A (I) and enzyme-bound phosphopantetheine (II) function as acyl carriers and as carbonyl activating groups for Claisen reactions as well as for amide-, ester-, and thioester-forming reactions in the cell. In so doing, these cofactors play a key role in the biosynthesis and breakdown of fatty acids and in the biosynthesis of polyketides and nonribosomal peptides. Coenzyme A is biosynthesized in bacteria in nine steps. The biosynthesis begins with the decarboxylation of aspartate to give ~-alanine. Pantoic acid is formed by the hydroxymethylation of ~-ketoisovalerate followed by reduction. These intermediates are then condensed to give pantothenic acid. Phosphorylation of pantothenic acid followed by condensation with cysteine and decarboxylation gives 4'-phosphopantetheine. Adenylation and phosphorylation of 4'-phosphopantetheine completes the biosynthesis ofcoenzyme A. This review will focus on the mechanistic enzymology of coenzyme A biosynthesis in bacteria. © 2001 Academic Press.

157 Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved.

0083-6729/01 $35.00

158 TADHG P. BEGLEY et al.

o SH

.b dP I

II

FIG. 1. The structure of eoenzyme A (I) and enzyme-bound phosphopantetheine (II).

I. INTRODUCTION

Coenzyme A (I) and enzyme-bound phosphopantetheine (II) (Fig. 1) function as acyl carriers and as carbonyl activating groups for Claisen reactions, as well as for amide-, ester-, and thioester-forming reactions in the cell. In so doing, these cofactors play a key role in the biosynthesis and breakdown of fatty acids and in the biosynthesis of nonribosomal peptides and polyketides. It has been estimated that 4% of the known enzymes use coenzyme A as a cofactor (Lee and Chen, 1982). This review will focus on the mechanistic enzymology of coenzyme A biosynthesis in bacteria.

I I . THE COENZYME A BIOSYNTHETIC PATHWAY IN BACTERIA

Bacteria synthesize coenzyme A from aspartate, ~-ketovalerate, cysteine, and ATP. The biosynthesis (Fig. 2) begins with the decarboxylation of aspartate (III) to give ~-alanine (IV). Pantoic acid (VII) is formed by the hydroxymethylation of a-ketoisovalerate (V) followed by reduction. These intermediates are then condensed to give pantothenic acid (VIII). Phosphorylation of pantothenic acid followed by condensation with cysteine and decarboxylation gives 4'-phosphopantetheine (XI). Adenylation and phosphorylation of 4'-phosphopantetheine completes the biosynthesis of coenzyme A. The acyl carrier protein is phosphopantetheinylated by the displacement of the AMP moiety of coenzyme A by an active site serine.

The biosynthesis of coenzyme A requires nine enzymes: aspartate l-decarboxylase, ketopantoate hydroxymethyltransferase, ketopantoate

BIOSYNTHESIS OF COENZYME A IN BACTERIA 159

Aspartate l-decarboxylase H N,~ ~COOH (PanD) -

2 H~,/COOH ,. H 2 N ~ C O O Aspartic acid ~3-Alanine

III IV Ketopantoate

reductase ~ . COOH /],~,.COO-hydroxymethyltransferase ( /~COO-Ketopantoa te

" ~ (PanB) (ApbA or PanE)" HO 0 NADPH H~) H" ~)H

ct-Ketoisovalerate c~-Ketopantoate Pantoic acid V Vl VII

Pantothenate ~ Pantothenatc O synthetase kinase "X~ ~. ~/L2OOH (PanC) H~ H4~'oHN/~"~OOH (CoaA) " ( H ~ I H N

PO ATP + IV Pantothenie acid 4'-Phosphopantothenie acid

VIII IX

4'-Phosphopantothenoyl- 4'-Phosphopantothenoylcysteine cysteine synthetase decarboxylase

(CoaB) (CoaC) , , .o. 9 H gOOH

Cysteine + CTP r /~I I" . - N "%"J~Y ~Q'/S H P6 ffb." H

4'-Phosphopantothenoylcysteine X

O 4'-Phosphopantetheine adenylyltransferase N / , . . ~ N ~ S H (CoaD)

-I H~bH H H ATP t'u 4'-Phosphopantetheine

XI

N

(~p.O O ] ' ' [ (CoaE) . _

"O"x~/ H6 (~H ATP ,~;\ , O O

H ~3H Coenzyme A

Dephospho Coenzyme A I XII

Phosphopantetheinyl transferase ~ S H ACP-Se

Acyl carrier protein-Ser-OH I1

FIG. 2. The coenzyme A biosynthetic pathway in bacteria.


reductase, pantothenate synthetase, pantothenate kinase, 4'-phosphopantothenoylcysteine synthetase, 4'-phosphopantothenoylcysteine decarboxylase, 4'-phosphopantetheine adenylyltransferase, and dephospho-coenzyme A kinase. The following sections review the progress made on the mechanistic enzymology of these enzymes during the 1990s (Jackowski, 1996).

A. ASPARTATE 1-DECARBOXYLASE

Aspartate 1-decarboxylase has been overexpressed from Escherichia coli (PanD, tetramer of 13.8-kDa subunits, K m = 151 txM, kca t = 0.6 s - 1 ) (Ramjee et al., 1997). This enzyme contains a pyruvamide cofactor that is formed by an autocatalytic cleavage between glycine 24 and serine 25 (Fig. 3). This cleavage reaction occurs by an N-O acyl shift to give XV followed by elimination and enamine hydrolysis. The autoprocess- ing reaction is quite slow (half-life of 16 h at 50°C), and the overexpressed enzyme after purification consists primarily of the unprocessed protein (90%). This raises the interesting possibility that an additional protein may be involved in the in vivo formation of the pyruvoyl cofactor because the time spent by E. coli between divisions (20-60 min) is much shorter than the in vitro cofactor processing time (Ramjee et al., 1997).

The crystal structure of aspartate 1-decarboxylase has been solved at 2.2 A resolution (Albert et al., 1998). In this structure, only three of the subunits contain the pyruvoyl cofactor, and the fourth subunit has been trapped as the ester intermediate (XV). The structure suggests that the

Gly 24 H H IOI O O N ~ N ~ LI'~ NHCyS26 n ~+r ~ " ~ NHCys26 H3~'~ ~U'~ NHCy s26 %-x,,, ) o1,%

O O

H 2 N ' ~ " NHCys26 ~ _ 0 ~ NHCys26

G I Y % o H [ [ XVII + NH3 I I O

XVl

FIG. 3. Mechanism for the formation of the pyruvoyl cofactor.


A subunit serine 25 is deprotonated by a proton relay system consist- ing oftyrosine 58 from the A subunit and lysine 9 and histidine 11 from the D subunit. The collapse of the resulting tetrahedral intermediate XIV is facilitated by the transfer of this proton from the relay to the amine via tyrosine 58. Either tyrosine 58 or threonine 57 may also function as the base catalyzing the elimination reaction (XV to XVI). In ad- dition, the structure also suggests that the N - O acyl shift (XIII to XV) and the elimination reaction (XV to XVI) are driven by relief of strain in a short peptide loop connecting [3 sheet 1' and [3 sheet 2 and by protonation of the amine XIV. The processing of the fourth subunit does not occur because of an unfavorable conformation for the elimination in this subunit and because there is no base close to the C proton of serine 25 to carry out the deprotonation reaction essential for the conver- sion of XV to XVI.

A mechanism for the decarboxylation reaction, based on mechanistic studies carried out on histidine decarboxylase, is outlined in Fig. 4 (Van Poelje and Snell, 1990). Formation of a protonated imine (XVIII) between aspar ta te and the pyruvoyl cofactor activates aspar ta te for decarboxylation because the resulting anion (XIX) is stabilized by an electrostatic interaction with the positively charged nitrogen and by de- localization (Bach and Canepa, 1997). Protonation of this anion followed by hydrolysis of the Schiffbase completes the reaction. The Schiff base of aspar ta te has been modeled into the active site of one of the fully processed subunits. This model suggests that aspar ta te is bound via a salt bridge to arginine A54 and that a water molecule hydrogen bonded to lysine A9 protonates the carbanion intermediate. Other in- teractions involved in the formation and hydrolysis of the Schiff base and[ in the decarboxylation have not yet been identified.

0 H

III / 0 XVII

0 O-

XVIII 0

O

H O O C ~ N H 2 , ~ H'Enz IV ~ 0 XVlI /

+ CO2

noocv-~n ~ H + CO 2

~" N. En z

XIX 0

FIG. 4. The mechanism of aspartate 1-decarboxylase.

162 TADHG R BEGLEYet al.

.R

H2 H

XX

-OOC~O _

v

H'J~H R

H XXIV

2 H2 H

XXI! XXIII

H ~H -00 H N +

~H2N H ~ O H XXI VI

FIG. 5. Mechanistic proposal for ketopantoate hydroxymethyltransferase.

B. KETOPANTOATE HYDROXYMETHYLTRANSFERASE

The gene coding for ketopantoate hydroxymethyltransferase in E. coli and in Aspergillus nidulans (panB) has been overexpressed (Jones et al., 1993; Kurtov et al., 1999; Merkel and Nichols, 1996). The purified E. coli enzyme is a hexamer of 28-kDa subunits and requires methylene tetrahydrofolate as a cosubstrate. The hydroxymethylation reaction is reversible and proceeds with inversion of stereochemistry at C-3 of 2-ketoisovalerate (Aberhart, 1979). A mechanistic proposal, based on the mechanism of serine hydroxymethyltransferase (Matthews and Drummond, 1990), is outlined in Fig. 5. In this mechanism, ring opening of methylene tetrahydrofolate (XX) followed by ad- dition of water to the resulting iminium ion gives XXIII. Extrusion of formaldehyde followed by its trapping by the enol of V would give ketopantoic acid VI.

C. KETOPANTOATE REDUCTASE

The ketopantoate reductase from E. coli has been cloned, overexpressed, and characterized [ApbA or PanE, monomer, 34 kDa, Km(NADPH) = 4.0 ~M, Km(VI) = 120 ~M, kca t = 8 s - l ] . The equilibri- um constant for the reaction is 676 in favor of VII, and the pro-(S) hydride of NADPH is transferred. The reaction proceeds by an ordered sequential kinetic mechanism in which NADPH binding is followed by ketopantoate binding. Isotope effect studies suggest that the hydride transfer step is not rate limiting (Zheng and Blanchard, 2000). Ke-


topantoic acid reductases from Salmonella typhimurium (Frodyma and Downs, 1998) and from Pseudomonas maltophilia (Shimizu et el., 1988) have also been characterized.

D. PANTOTHENATE SYNTHASE

Although the gene for pantothenate synthase (panC) has been iden- tiffed, it has not been overexpressed from any bacterial source. The E. coli enzyme has been purified 500-fold and is a homotetramer of 18-kDa subunits. The K m values for ~-alanine, ATP, and pantoic acid are 150, 100, and 63 txM, respectively. The enzyme follows a Ping-Pong Bi Uni Bi type mechanism (Miyatake et el., 1979).

Pantothenate is the most advanced coenzyme A precursor that is taken up by E. coli. The transport protein, pantothenate permease (PanF), has been cloned. Uptake occurs by a sodium ion-dependent cotransport mechanism (Jackowski and Alix, 1990).

The pantothenate synthetase genes from Lotus japonicus and from Oryza sat ivum (rice) have also been cloned (Genschel et el., 1999). The Lotusjaponicus enzyme has been overexpressed and characterized [homodimer of 34-kDa subunits, KIn(VII) = 44 ~M, Kin(IV) = 44 ~M, hea t = 0.62 s-l) .

E. PANTOTHENATE KINASE

Pantothenate kinase has been cloned from E. coli and characterized [CoaA homodimer, Km(ATP) = 136 txM, KIn(VIII) = 36 IxM, k c a t = 17 rain 1) (Song and Jackowski, 1992). The enzyme is expressed as two closely related translation products (35.4 and 36.4 kDa). The signifi- cance of this observation is unknown. The phosphorylation reaction proceeds by an ordered sequential mechanism with ATP binding first. The enzyme is inhibited by coenzyme A and its esters, shows highly co- operative ATP binding, and is a key regulatory enzyme on the coenzyme A biosynthesis pathway (Song and Jackowski, 1994). Pantothenate kinase has been overexpressed and characterized from AspergiUus nidu- lens (Calder et el., 1999), and the enzyme has also been purified from wild-type Brevibacterium ammoniagenes (Shimizu et el., 1973).

F. 4%PHosPHOPANTOTHENOYLCYSTEINE SYNTHASE

4'-Phosphopantothenoylcysteine synthase activity has been de- tected in a cell-free system, but the enzyme has not been purified from


any bacterial source (Brown, 1959). The gene coding for this enzyme has not been identified.

G. 4 ' -PHosPHOPANTOTHENOYLCYSTEINE DECARBOXYLASE

4'-Phosphopantothenoylcysteine decarboxylase has been purified from E. coli (homotetramer of 35-kDa subunits, K m = 0.9 mM, k c a t =

21 min -1 assuming one active site per subunit) (Yang and Abeles, 1987). This enzyme utilizes a pyruvoyl group and is mechanistically interesting because all other pyruvoyl-dependent enzymes require a free amino group a to the carboxylate in order to catalyze the decarboxylation (see aspartate 1-decarboxylase, Fig. 4). No mechanistic studies have been carried out on this enzyme other than the demonstration that the decarboxylation catalyzed by the rat liver enzyme occurs with re- tention of stereochemistry (Aberhart et al., 1985). The gene has not yet been identified in any microorganism.

A mechanistic proposal for this interesting decarboxylase is outlined in Fig. 6. In this mechanism an N-S acyl shift unmasks the amino group to give XXVI. The free amine could then participate in a pyruvoyl-assisted decarboxylation as previously described in Fig. 4 for aspartate decarboxylase. Reversal of the N-S acyl shift would complete the reaction. Enzyme-catalyzed acyl shift reactions have been previously identified in the intein processing reaction (Paulus, 1998; Perler, 1998; Perler et al., 1997; Shao and Paulus, 1997), in the formation of

0 OH

y "-00- N - - ' ( • y "OH2N % 0 N

( R _/S | S ENZYME ENZYME ] ] ENZYME R ~

XXV X XXV 0 XXV] XXV[I 0

| R ~S / HS ENZYME - '~ ENZYME " ~ ENZYME

0 0 XXVIII XXV XXIX XXV XI

FIG. 6. Proposed mechanism for 4'-phosphopantothenoylcysteine decarboxylase (R = 4'-phosphopantothenoyl).


the pyruvoyl cofactor (Van Poelje and Snell, 1990), and in the serine and cysteine proteases.

H. 4'-PHosPHOPANTETHEINE ADENYLYLTRANSFERASE

Tihe 4'-phosphopantetheine adenylyltransferase gene (kdtB or coaD) has been overexpressed and purified from E. coli (Geerlofet al., 1999). It is a hexamer of 17.8-kDa subunits. The adenylation reaction is reversible [Km(XI) = 7 ~M, Km(PP i) = 0.2 raM, kca t =- 3.3 s-l]. The crystal structure of this enzyme complexed with dephospho-coenzyme A has been described (Izard and Geerlof, 1999).

I. DEPHOSPHO-COENZYME A KINASE

The E. coli dephospho-coenzyme A kinase gene has recently been identified, and the enzyme has been overexpressed and characterized (D. Drueckhammer, unpublished results, 2000).

4'-Phosphopantetheine adenylyltransferase and dephospho-coen- zyrae A kinase have been used for the enzymatic synthesis of coenzyme A analogs (Bibart et al., 1999; Martin et al., 1994). These analogs have been elegantly used as mechanistic probes of coenzyme A-utilizing enzymes (Gu et al., 1999; Kurz et al., 1997; Schwartz and Drueckhammer, 1996; Schwartz et al., 1995; Usher et al., 1994).

J. 4'-PHosPHOPANTETHEINYLTRANSFERASE

The phosphopantetheinyl moiety of the acyl carrier protein is formed by displacement of AMP from coenzyme Aby an active site serine in a reaction catalyzed by a phosphopantetheinyltransferase. The enzyme has been overexpressed and characterized from several sources. These include the acyl carrier protein synthase from E. coli involved in fatty acid biosynthesis (Flugel et al., 2000), Lys5 from Sac- charomyces cerevisiae involved in lysine biosynthesis (Ehmann et al., 1999), EntD from E. coli involved in enterobactin biosynthesis (Gehring et al., 1998), and Spf from B. subt i l is involved in surfactin biosynthesis (Quadri et al., 1998). The Spf phosphopantetheinyltransferase shows relaxed substrate specificity and has been used to transfer the phosphopantetheinyl moiety to apo-6-methylsalicylic acid synthase in S. cerevisiae and in E. coli, giving rise to active synthase and the overproduction of 6-methylsalicylic acid (Carreras et al., 1997; Kealey et al., 1998).


III. REGULATION OF COENZYME A BIOSYNTHESIS IN Escherichia coli

Pantothenic acid is not a rate-limiting intermediate because E. coli produces 15 times more pantothenate than is required for coenzyme A biosynthesis. Furthermore, the overexpression of pantothenate permease in the presence ofpantothenate does not result in an increase in the concentration of coenzyme A in the cell (Jackowski and Rock, 1981; Jackowski and Alix, 1990).

Pantothenate kinase is a key control point on the coenzyme A biosynthesis pathway. It is subject to feedback inhibition by coenzyme A and to a lesser extent by coenzyme A thioesters (Vallari et al., 1987; Song and Jackowski, 1994). As a consequence, 76-fold overexpression of pantothenate kinase resulted in only a 2.7-fold increase in the concentration of cellular coenzyme A (Song and Jackowski, 1992). The concentration of coenzyme A in the cell is also influenced by coenzyme A degradation (Vallari and Jackowski, 1988). This degradation reaction is not catalyzed by phosphopantetheine adenylyltransferase (Geerlofet al., 1999), and the enzyme involved has not yet been identified.

IV. COENZYME A BIOSYNTHESIS IN Saccharomyces cerevisiae

In Saccharomyces cerevisiae pantothenate is t ransported into the cell by the Fen2p transporter (Stolz and Sauer, 1999) and converted to coenzyme A by a 375- to 400-kDa multienzyme complex (Bucovaz et al., 1997). The biosynthetic pathway (Fig. 7) uses essentially the same chemistry as that found in the bacterial pathway although the steps occur in a different order. Pantothenate (VIII) is first adenylylated and then condensed with cysteine to give XXXI. Decarboxylation followed by a final phosphorylation completes the biosynthesis. Our understanding of the mechanistic enzymology of coenzyme A biosynthesis in yeast is still at an early stage.

V. COENZYME A IN HUMAN NUTRITION

Pantothenic acid (vitamin B 5) is the most advanced coenzyme A precursor taken up by cells. It is not biosynthesized by humans and is, therefore, an essential vitamin. Annual production of pantothenic acid is 5000 tons, and the average daily intake, primarily in the form ofcoenzyme A and coenzyme A esters, is 5 -10 mg per person. A recommended

VIII

BIOSYNTHESIS OF COENZYME A IN BACTERIA

~.O_P/=O HO 0tl

167

N

-o-pL = o o

XXXI

O•P 0 HO OH

XI1 H ~'OH 1

F I a . 7. C o e n z y m e A b i o s y n t h e s i s i n Saccharomyces cerevisiae.

dietary allowance has not been established because this vitamin is widely distributed in plants and animals and because spontaneous pantothenic acid deficiency in humans has not been recorded. Dietary coenzyme A is hydrolyzed in the lumen of the intestine to pantothenic acid, which is then taken up and reconverted to coenzyme A (Brody, 1998; Tahiliani and Beinlich, 1991). Our current understanding of the mechanistic enzymology of coenzyme A biosynthesis in mammals is still at an early stage.

VI. SUMMARY AND CONCLUSIONS

Bacteria biosynthesize coenzyme A from aspartate, ~-ketovalerate, cysteine, and ATP. The biosynthesis begins with the decarboxylation of aspartate (III) to give ~-alanine (IV) and the formation ofpantoic acid (VII) by the hydroxymethylation of ~-ketoisovalerate (V) followed by reduction. These intermediates are then condensed to give pantothenic acid (VIII). Phosphorylation of pantothenic acid followed by condensation with cysteine and decarboxylation gives phosphopantetheine (XI). Adenylation and phosphorylation of this complete the biosynthesis of coenzyme A. Overall, the coenzyme A biosynthesis pathway in-


volves well-understood chemistry except for the decarboxylation of phosphopantothenoylcysteine. This reaction is a novel pyruvoyl- dependent decarboxylation reaction in which the substrate does not have a free amino group. All of the biosynthetic genes except for the phosphopantothenoylcysteine synthetase gene and the phosphopantothenoylcysteine decarboxylase gene have been identified. Structural studies on the pathway are at an early stage, and only two structures have been solved (aspartate decarboxylase and 4'-phosphopantetheine adenylyltransferase). The mechanistic enzymology of coenzyme A biosynthesis in eukaryotes is also at an early stage.

REFERENCES

Aberhart, D. J. (1979). Stereochemistry of pantoate biosynthesis from 2-ketoisovalerate. J. Am. Chem. Soc. 101, 1354-1355.

Aberhart, D. J., Ghoshal, P. K., Cotting, J.-A., and Russell, D. J. (1985). Coenzyme A biosynthesis: Steric course of4'-phosphopantothenoyl-L-cysteine decarboxylase. Bio- chemistry 24, 7178-7182.

Albert, A., Dhanaraj, V., Genschel, U., Khan, G., Ramjee, M. K., Pulido, R., Sibanda, B. L., von Delft, F., Witty, M., Blundell, T. L., Smith, A. G., and Abell, C. (1998). Crystal structure of aspartate decarboxylase at 2.2 A resolution provides evidence for an ester in protein self-processing. Nat. Struct. Biol. 5, 289-293.

Bach, R. D., and Canepa, C. (1997). Theoretical model for pyruvoyl-dependent enzymic decarboxylation of a-amino acids. J. Am. Chem. Soc. 119, 11725-11733.

Bibart, R. T., Vogel, K. W., and Drueckhammer, D. G. (1999). Development of a second generation coenzyme A analog synthon. J. Org. Chem. 64, 2903-2909.

Brody, T. (1998). "Nutritional Biochemistry," 2nd ed. Academic Press, San Diego, CA. Brown, G. M. (1959). The metabolism of pantothenic acid. J. Biol. Chem. 234, 370-378. Bucovaz, E. T., Macleod, R. M., Morrison, J. C., and Whybrew, W. D. (1997). The coen-

zyme A synthesizing protein complex and its proposed role in CoA biosynthesis in bakers' yeast. Biochimie 79, 787-798.

Calder, R. B., Williams, R. S., Ramaswamy, G., Rock, C. O., Campbell, E., Unkles, S. E., Kinghorn, J. R., and Jackowski, S. (1999). Cloning and characterization of a eukaryotic pantothenate kinase gene (panK) from Aspergillus nidulans. J. Biol. Chem. 274, 2014-2020.

Carreras, C. W., Gehring, A. M., Walsh, C. T., and Khosla, C. (1997). Utilization of en- zymically phosphopantetheinylated acyl carrier proteins and acetyl-acyl carrier proteins by the actinorhodin polyketide synthase. Biochemistry 36, 11757-11761.

Ehmann, D. E., Gehring, A. M., and Walsh, C. T. (1999). Lysine biosynthesis in Saccha- romyces cerevisiae: Mechanism of ~-aminoadipate reductase (Lys2) involves post- translational phosphopantetheinylation by Lys5. Biochemistry 38, 6171-6177.

Flugel, R. S., Hwangbo, Y., Lambalot, R. H., Cronan, J. E., and Walsh, C. T. (2000). Holo- (acyl carrier protein) synthase and phosphopantetheinyl transfer in Escherichia coli. J. Biol. Chem. 275, 959-968.

Frodyma, M. E., and Downs, D. (1998). ApbA, the ketopantoate reductase enzyme of Sal- monella typhimurium, is required for the synthesis of thiamine via the alternative pyrimidine biosynthetic pathway. J. Biol. Chem. 273, 5572-5576.


Geerlof, A., Lewendon, A., and Shaw, W. V. (1999). Purification and characterization of phosphopantetheine adenylyltransferase from Escherichia coll. J. Biol. Chem. 274, 2'7105-27111.

Gehring, A. M., Mori, I., and Walsh, C. T. (1998). Reconstitution and characterization of tile Escherichia coli enterobactin synthetase from EntB, EntE, and EntF. Biochem- istry 37, 2648-2659.

Genschel, U., Powell, C. A., Abell, C., and Smith, A. G. (1999). The final step of pantothenate biosynthesis in higher plants: Cloning and characterization of pantothenate synthetase from Lotusjaponicus and Oryza sativum (rice). Biochem. J. 341,669- 678.

Gu, Z, Drueckhammer, D. G., Kurz, L., Liu, K., Martin, D. P., and McDermott, A. (1999). Solid state NMR studies of hydrogen bonding in a citrate synthase inhibitor complex. Biochemistry 38, 8022-8031.

Izard: T., and Geerlof, A. (1999). The crystal structure of a novel bacterial adenylyltransferase reveals half of sites reactivity. EMBO J. 18, 2021-2030.

Jackowski, S. (1996). Biosynthesis of pantothenic acid and coenzyme A. In "Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology" (F. C. Neidhardt et al., eds.), 2nd ed., pp. 687-694. American Society for Microbiology, Washington, DC.

Jackowski, S., and Alix, J. H. (1990). Cloning, sequence and expression of the pantothenate permease (PanF) gene of Escherichia coli. J. Bacteriol. 172, 3842-3848.

Jackowski, S., and Rock, C. O. (1981). Regulation of coenzyme A biosynthesis. J. Bacte- rliol. 148, 926-932.

Jones;, C. E., Brook, J. M., Buck, D., Abell, C., and Smith, A. G. (1993). Cloning and sequencing of the Escherichia coli panB gene, which encodes ketopantoate hydroxymethyltransferase, and overexpression of the enzyme. J. Bacteriol. 175, 2125- 2130.

Kealey, J. T., Liu, L., Santi, D. V., Betlach, M. C., and Barr, P. J. (1998). Production of a polyketide natural product in nonpolyketide-producing prokaryotic and eukaryotic hosts. Proc. Natl. Acad. Sci. U.S.A. 95, 505-509.

Kurtov, D., Kinghorn, J. R., and Unkles, S. E. (1999). The Aspergillus nidulans panB gene encodes ketopantoate hydroxymethyltransferase, required for biosynthesis of pantothenate and coenzyme A. Mol. Gen. Genet. 262, 115-120.

Kurz, L. C., Roble, J. H., Nakra, T., Drysdale, G. R., Buzan, J. M., Schwartz, B., and Drueckhammer, D. G. (1997). Ability of single-site mutants of citrate syuthase to catalyze proton transfer from the methyl group of dethiaacetyl-coenzyme A, a non- thioester substrate analog. Biochemistry 36, 3981-3990.

Lee, C. H., and Chen, A. F. (1982). Immobilized coenzymes and derivatives. In "The Pyri- dine Nucleotide Coenzymes" (J. Everse, B. Anderson, and K. You, eds.), pp. 189. Aca- demic Press, New York.

Martin, D. P., Bibart, R. T., and Drueckhammer, D. G. (1994). Synthesis of novel analogs of acetyl coenzyme A: Mimics of enzyme reaction intermediates. J. Am. Chem. Soc. 116, 4660-4668.

Matthews, R. G., and Drummond, J. T. (1990). Providing one-carbon units for biological methylations: Mechanistic studies on serine hydroxymethyltransferase, methyl- enetetrahydrofolate reductase, and methyltetrahydrofolate-homocysteine methyltransferase. Chem. Rev. 90, 1275-1290.

Merkel, W. K., and Nichols, B. P. (1996). Characterization and sequence of the Escherichia coli panBCD gene cluster. FEMB Microbiol. Lett. 143, 247-252.


Miyatake, K., Nakano, Y., and Kitaoka, S. (1979). Pantothenate synthetase from Es- cherichia coli. In "Methods in Enzymology" (D. B. McCormick and L. D. Wright, eds.), Vol. 62, Part D, pp. 215-219. Academic Press, New York.

Paulus, H. (1998). The chemical basis of protein splicing. Chem. Soc. Rev. 27, 375-386. Perler, F. B. (1998). Breaking up is easy with esters. Nat. Struct. Biol. 5, 249-252. Perler, F. B., Xu, M.-Q., and Paulus, H. (1997). Protein splicing and autoproteolysis mech-

anisms. Curr. Opin. Chem. Biol. 1, 292-299. Quadri, L. E. N., Weinreb, P. H., Lei, M., Nakano, M. M., Zuber, P., and Walsh, C. T.

(1998). Characterization of Sfp, a Bacillus subtilis phosphopantetheinyl transferase for peptidyl carrier protein domains in peptide synthetases. Biochemistry 37, 1585- 1595.

Ramjee, M. K., Genschel, U., Abell, C., and Smith, A. G. (1997). Escherichia coli L-aspartate-(~-decarboxylase: Preprotein processing and observation of reaction intermediates by electrospray mass spectrometry. Biochem. J. 323, 661-669.

Schwartz, B., and Drueckhammer, D. G. (1996), A stereochemical probe of the tetrahedral intermediate in the reactions of acetyl-coenzyme A dependent acetyltransferas- es. J. Am. Chem. Soc. 118, 9826-9830.

Schwartz, B., Drueckhammer, D. G., Usher, K. C., and Remington, S. J. (1995). ~-Fluoro acid and ~-fluoro amide analogs of acetyl-CoA as inhibitors of citrate synthase: Ef- fect o fpK matching on binding affinity and hydrogen bond length. Biochemistry 34, 15459-15466.

Shao, Y., and Paulus, H. (1997). Protein splicing: Estimation of the rate of O-N and S- N acyl rearrangements, the last step of the splicing process. J. Pept. Res. 50, 193- 198.

Shimizu, S., Kubo, K., Tani, Y., and Ogata, K. (1973). Metabolism of pantothenic acid in microorganisms. VII. Purification and properties of pantothenate kinase from Bre- vibacterium ammoniagenes IFO 12071. Agric. Biol. Chem. 37, 2863-2870.

Shimizu, S., Kataoka, M., Chung, M., and Yamada, H. (1988). Ketopantoic acid reductase of Pseudomonas maltophilia 845. Purification, characterization, and role in pantothenate biosynthesis. J. Biol. Chem. 263, 12077-12084.

Song, W.-J., and Jackowski, S. (1992). Cloning, sequencing, and expression of the pantothenate kinase (coaA ) gene of Escherichia coli. J. Bacteriol. 174, 6411-6417.

Song, W.-J., and Jackowski, S. (1994). Kinetics and regulation of pantothenate kinase from Escherichia coli. J. Biol. Chem, 269, 27051-27058.

Stolz, J., and Sauer, N. (1999). The fenpropimorph resistance gene FEN2 from Saccha- romyces cerevisiae encodes a plasma membrane H÷-pantothenate symporter. J. Biol. Chem. 274, 18747-18752.

Tahiliani, A. G., and Beinlich, C. J. (1991). Pantothenic acid in health and disease. Vitam. Horm. (N.Y,)46, 165-228.

Usher, K. C., Remington, S. J., Martin, D. P., and Drueckhammer, D. G. (1994). A very short hydrogen bond provides only moderate stabilization of an enzyme-inhibitor complex of citrate synthase. Biochemistry 33, 7753-7759.

Vallari, D. S., and Jackowski, S. (1988). Biosynthesis and degradation both contribute to the regulation of coenzyme A content in Escherichia coli. J. Bacteriol. 170, 3961.

Vallari, D. S., Jackowski, S., and Rock, C. O. (1987). Regulation of pantothenate kinase by coenzyme A and its thioesters. J. Biol. Chem. 262, 2468-2471.

Van Poelje, P. D., and Snell, E. E. (1990). Pyruvoyl-dependent enzymes. Annu, Rev. Biochem. 59, 29-59.

Yang, H., and Abeles, R. H. (1987). Purification and properties ofEscherichia coli 4'-phos-


phopantothenoylcysteine decarboxylase: Presence of covalently bound pyruvate. Bio- chemistry 26, 4076-4081.

Zheng, R., and Blanchard, J. S. (2000). Kinetic and mechanistic analysis of the E. coli pan E-.encoded ketopantoate reductase. Biochemistry 39, 3708-3717.

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The biosynthesis of coenzyme A in bacteria