X-ray structure of pyruvate formate-lyase in complex with pyruvate and CoA. How the enzyme uses the Cys-418 thiyl radical for pyruvate cleavage

The glycyl radical enzyme pyruvate formate-lyase (PFL) synthesizes acetyl-CoA and formate from pyruvate and CoA. With the crystal structure of the non-radical form of PFL in complex with its two substrates, we have trapped the moment prior to pyruvate cleavage. The structure reveals how the active site aligns the scissile bond of pyruvate for radical attack, prevents non-radical side reactions of the pyruvate, and confines radical migration. The structure shows CoA in a syn conformation awaiting pyruvate cleavage. By changing to an anti conformation, without affecting the adenine binding mode of CoA, the thiol of CoA could pick up the acetyl group resulting from pyruvate cleavage.     

Structure and mechanism of the glycyl radical enzyme pyruvate formate-lyase.

Pyruvate formate-lyase (PFL) from Escherichia coli uses a radical mechanism to reversibly cleave the C1-C2 bond of pyruvate using the Gly 734 radical and two cysteine residues (Cys 418, Cys 419). We have determined by X-ray crystallography the structures of PFL (non-radical form), its complex with the substrate analog oxamate, and the C418A,C419A double mutant. The atomic model (a dimer of 759-residue monomers) comprises a 10-stranded beta/alpha barrel assembled in an antiparallel manner from two parallel five-stranded beta-sheets; this architecture resembles that of ribonucleotide reductases. Gly 734 and Cys 419, positioned at the tips of opposing hairpin loops, meet in the apolar barrel center (Calpha-Sgamma = 3.7 A). Oxamate fits into a compact pocket where C2 is juxtaposed with Cys 418Sgamma (3.3 A), which in turn is close to Cys 419Sgamma (3.7 A). Our model of the active site is suggestive of a snapshot of the catalytic cycle, when the pyruvate-carbonyl awaits attack by the Cys 418 thiyl radical. We propose a homolytic radical mechanism for PFL that involves Cys 418 and Cys 419 both as thiyl radicals, with distinct chemical functions.     

Purification of pyruvate formate-lyase from Streptococcus mutans and its regulatory properties.

Pyruvate formate-lyase (EC 2.3.1.54) from Streptococcus mutans strain JC2 was purified in an anaerobic glove box, giving a single band on disk and sodium dodecyl sulfate electrophoresis. This enzyme was immediately inactivated by exposure to the air. Enzyme activity was unstable even when stored anaerobically, but the activity was restored by preincubating the inactivated crude enzyme with S-adenosyl-L-methionine, oxamate, and reduced for ferredoxin or methylviologen. On the other hand, the purified enzyme was not reactivated. Either D-glyceraldehyde 3-phosphate or dihydroxyacetone phosphate strongly inhibited this enzyme. The inhibitory effects of these compounds were largely influenced by enzyme concentration. The inhibition of these triose phosphates in cooperation with the reactivating effect of ferredoxin and the fluctuations of both the enzyme and the triose phosphate levels may efficiently regulate the pyruvate formate-lyase activity in S. mutans in vivo.     

Ultrastructure and pyruvate formate-lyase radical quenching property of the multienzymic AdhE protein of Escherichia coli.

The AdhE protein of Escherichia coli is a homopolymer of 96-kDa subunits harboring three Fe(2+)-dependent catalytic functions: acetaldehyde-CoA dehydrogenase, alcohol dehydrogenase, and pyruvate formatelyase (PFL) deactivase. By negative staining electron microscopy, we determined a helical assembly of 20-60 subunits into rods of 45-120 nm in length. The subunit packing is widened along the helix axis when Fe2+ and NAD are present. Chymotrypsin dissects the AdhE polypeptide between Phe762 and Ser763, thereby retaining the alcohol dehydrogenase activity on the NH2-terminal core, but destroying all other activities. PFL deactivation, i.e. quenching of the glycyl radical in PFL by the AdhE protein, was examined with respect to cofactor involvements (Fe2+, NAD, and CoA). This process is coupled to NAD reduction and requires the intact CoA sulfhydryl group. Pyruvate and NADH are inhibitors that affect the steady-state level of the radical form of PFL in a reconstituted interconversion cycle. Studies of cell cultures found that PFL deactivation in situ is initiated at redox potentials of greater than or equal to +100 mV. Our results provide insights into the structure/function organization of the AdhE multienzyme and give a rationale for how its PFL radical quenching activity may be suppressed in situ to enable effective glucose fermentation.     

Rescuing activity of oxygen-damaged pyruvate formate-lyase by a spare part protein

Pyruvate formate-lyase (PFL) is a glycyl radical enzyme (GRE) that converts pyruvate and coenzyme A into acetyl-CoA and formate in a reaction that is crucial to the primary metabolism of many anaerobic bacteria. The glycyl radical cofactor, which is posttranslationally installed by a radical S-adenosyl-L-methionine (SAM) activase, is a simple and effective catalyst, but is also susceptible to oxidative damage in microaerobic environments. Such damage occurs at the glycyl radical cofactor, resulting in cleaved PFL (cPFL). Bacteria have evolved a spare part protein termed YfiD that can be used to repair cPFL. Previously, we obtained a structure of YfiD by NMR spectroscopy and found that the N-terminus of YfiD was disordered and that the C-terminus of YfiD duplicates the structure of the C-terminus of PFL, including a β-strand that is not removed by the oxygen-induced cleavage. We also showed that cPFL is highly susceptible to proteolysis, suggesting that YfiD rescue of cPFL competes with protein degradation. Here, we probe the mechanism by which YfiD can bind and restore activity to cPFL through enzymatic and spectroscopic studies. Our data show that the disordered N-terminal region of YfiD is important for YfiD glycyl radical installation but not for catalysis, and that the duplicate β-strand does not need to be cleaved from cPFL for YfiD to bind. In fact, truncation of this PFL region prevents YfiD rescue. Collectively our data suggest the molecular mechanisms by which YfiD activation is precluded both when PFL is not damaged and when it is highly damaged.     

The free radical of pyruvate formate-lyase. Characterization by EPR spectroscopy and involvement in catalysis as studied with the substrate-analogue hypophosphite

The first-derivative EPR spectrum of the active form of Escherichia coli pyruvate formate-lyase shows an asymmetric doublet with partially resolved hyperfine splittings (g = 2.0037). Isotope substitution studies demonstrated couplings of a carbon-centered unpaired electron to a solvent-exchangeable proton (a = 1.5 mT) and to further hydrogen nuclei (a = 0.36 and 0.57 mT). By selective incorporation of unlabelled tyrosine into 2H-labelled enzyme protein, a tyrosyl radical structure has been ruled out. Circumstantial evidence indicates that the organic free radical, which also displays an ultraviolet absorption signal at 365 nm, is located on a standard amino acid residue of the polypeptide chain. EPR signal quantification found a stoichiometry of 1 spin per active site. The formate analogue hypophosphite has been characterized as a specific kcat inhibitor of pyruvate formate-lyase which destroys the enzyme radical. Protein-linked 1-hydroxyethylphosphonate was previously described as the dead-end product after reaction of the analogue with the intermediary acetyl-enzyme form of the catalytic cycle [W. Plaga et al. (1988) Eur. J. Biochem. 178, 445-450]. EPR spectroscopy of this system has now identified the corresponding alpha-phosphoryl radical as a reaction intermediate [g = 2.0032; a(P) = 2.72 mT, a(3H) = 1.96 mT]; it showed a half-life of about 20 min at 0 degrees C. This finding proves that the enzyme radical is a hydrogen-atom-transferring coenzymic element.     

Pyruvate formate-lyase activating enzyme: elucidation of a novel mechanism for glycyl radical formation

Pyruvate formate lyase activating enzyme is a member of a novel superfamily of enzymes that utilize S-adenosylmethionine to initiate radical catalysis. This enzyme has been isolated with several different iron-sulfur clusters, but single turnover monitored by EPR has identified the [4Fe-4S](1+) cluster as the catalytically active cluster; this cluster is believed to be oxidized to the [4Fe-4S](2+) state during turnover. The [4Fe-4S] cluster is coordinated by a three-cysteine motif common to the radical/S-adenosylmethionine superfamily, suggesting the presence of a unique iron in the cluster. The unique iron site has been confirmed by Mossbauer and ENDOR spectroscopy experiments, which also provided the first evidence for direct coordination of S-adenosylmethionine to an iron-sulfur cluster, in this case the unique iron of the [4Fe-4S] cluster. Coordination to the unique iron anchors the S-adenosylmethionine in the active site, and allows for a close association between the sulfonium of S-adenosylmethionine and the cluster as observed by ENDOR spectroscopy. The evidence to date leads to a mechanistic proposal involving inner-sphere electron transfer from the cluster to the sulfonium of S-adenosylmethionine, followed by or concomitant with C-S bond homolysis to produce a 5'-deoxyadenosyl radical; this transient radical abstracts a hydrogen atom from G734 to activate pyruvate formate lyase.     

Computer-aided design of the stability of pyruvate formate-lyase from Escherichia coli by site-directed mutagenesis

Using computer-aided design of single-site mutations, three amino acid residues determined by changes in folding free energy between wild-type (wt) and mutant proteins were exchanged to enhance the stability of pyruvate formate-lyase (PFL). The mutant enzymes were tested for properties such as optimum temperature, optimum pH, kinetic parameters, and stability to temperature. There were two mutant variants, Glu336Cys and Glu400Ile, that exhibited increased thermostability as compared to the wt enzyme. The melting temperatures (T(m), the temperature at which 50% inactivation occurs after heat treatment for 20 min) of Glu336Cys and Glu400Ile increased by 3.7 and 2.2 respectively. They also showed an increase in half life of about 1.80 and 2.21-fold, whereas Ala273Cys showed a slight decrease as compared with the wt enzyme.     

Involvement of oxygen-sensitive pyruvate formate-lyase in mixed-acid fermentation by Streptococcus mutans under strictly anaerobic conditions

Streptococcus mutans JC2 produced formate, acetate, ethanol, and lactate when suspensions were incubated with an excess of galactose or mannitol under strictly anaerobic conditions. The galactose- or mannitol-grown cell suspensions produced more formate, acetate, and ethanol than the glucose-grown cells even when incubated with glucose. The levels of lactate dehydrogenase and fructose 1,6-bisphosphate were not significantly different in these cells, but the level of pyruvate formate-lyase was higher in the galactose- or mannitol-grown cells, and that of triose phosphate was lower in the galactose-grown cells. This suggests that the regulation of pyruvate formate-lyase may play a major role in the change of the fermentation patterns. The cells of S. mutans grown on glucose produced a significant amount of volatile products even in the presence of excess glucose under strictly anaerobic conditions. However, when the anaerobically grown cells were exposed to air, only lactate was produced from glucose. When cells were anaerobically grown on mannitol and then exposed to air for 2 min, only trace amounts of fermentation products were formed from mannitol under anaerobic conditions. It was found that the pyruvate formate-lyase in the cells was inactivated by exposure of the cells to air.     

Inactivation of Escherichia coli pyruvate formate-lyase by hypophosphite: evidence for a rate-limiting phosphorus-hydrogen bond cleavage

Recently, Knappe and co-workers [Knappe, J., Neugebauer, F. A., Blaschkowski, H. P., & Ganzler, M. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1332] have shown that the catalytically active form of pyruvate formate-lyase from Escherichia coli is associated with a protein-bound organic free radical which is quenched upon enzyme inactivation by oxygen or hypophosphite. Our interest in the chemical mechanism of this unusual enzymatic reaction has led us to investigate several key aspects of the inactivation of the lyase by hypophosphite and its relationship to the normal enzymatic reaction. We report here that the inactivation of both the free and acetylated forms of the lyase is subject to a primary kinetic isotope effect using [2H2]hypophosphite. This suggests that phosphorus-hydrogen bond cleavage is at least partially rate limiting during inactivation. In addition, the inactivated enzyme can be fully reactivated. We have also determined a Vmax/Km isotope effect of 3.6 +/- 0.7 for pyruvate formation from [2H]formate and acetyl coenzyme A. Thus, carbon-hydrogen bond cleavage is partially rate limiting in the normal reverse reaction. On the basis of our findings, the previous work of Knappe and co-workers, the likelihood that hypophosphite is a formate analogue, the known susceptibility of both hypophosphite and formate to homolysis, and a chemical precedent for homolytic cleavage of pyruvate, we offer a preliminary mechanistic proposal for the lyase reaction.     

High production of D-β-hydroxyisobutyric acid from methacrylic acid by Candida rugosa and its mutant

Production of D-β-hydroxyisobutyric acid (D-HIBA) from methacrylic acid (MA) was investigated using Candida rugosa IFO 0750 and its mutant. Cell growth decreased as the MA concentration increased and was inhibited at D-HIBA concentrations higher than 30 g/l. Optimal MA concentration for D-HIBA production was in the range of 10–20 g/l. It was also noted that cell growth and D-HIBA production were inhibited by higher concen-trations of Na⁺, K⁺, and NH₄ ⁺, which were required for pH control during cultivation. With a suitably designed feeding mode of MA, the parent strain produced 65 g/l of D-HIBA after 120?h of fed-batch cultivation, but molar conversion yield of D-HIBA was less than 40%. The mutant, unable to assimilate propionic acid, produced as high as 70 g/l of D-HIBA in the same culture period with a molar conversion yield of more than 70%.     

Modification of bovine kidney pyruvate dehydrogenase kinase activity by CoA esters and their mechanism of action

The activation of pyruvate dehydrogenasea kinase activity by CoA esters has been further characterized. Half-maximal activation of kinase activity was achieved with about 1.0 microM acetyl-CoA after a 20-s preincubation in the presence of NADH. More than 80% of the acetyl-CoA was consumed during this period in acetylating sites in the pyruvate dehydrogenase complex as a result of the transacetylation reaction proceeding to equilibrium. At 1.0 microM acetyl-CoA, this resulted in more than a 4-fold higher level of CoA than residual acetyl-CoA. Activation of kinase activity could result either from acetylation of specific sites in the complex or tight binding of acetyl-CoA. Removal of CoA enhanced both acetylation and activation, suggesting acetylation mediates activation. For allosteric binding of acetyl-CoA to elicit activation, an activation constant, Ka, less than 50 nM would be required. To further distinguish between those mechanisms, the effects of other CoA esters as well as the reactivity of most of the effective CoA esters were characterized. Several short-chain CoA esters enhanced kinase activity including (in decreasing order of effectiveness) malonyl-CoA, acetoacetyl-CoA, propionyl-CoA, and methylmalonyl-CoA. Butyryl-CoA inhibited kinase activity as did high concentrations of long-chain acyl-CoAs. Inhibition by long-chain acyl-CoAs may result, in part, from detergent-like properties of those esters. Malonyl-CoA, propionyl-CoA, butyryl-CoA, and methylmalonyl-CoA, obtained with radiolabeled acyl groups, were shown to acylate sites in the complex. Propionyl-CoA and butyryl-CoA were tested, in competition with acetyl-CoA or pyruvate, as alternative substrates for acylation of sites in the complex and as competitive effectors of kinase activity. Propionyl-CoA alone rapidly acylated sites in the complex at low concentrations, and low concentrations of propionyl-CoA were effective in activating kinase activity although only a relatively small activation was observed. When an equivalent level (20 microM) of acetyl-CoA and propionyl-CoA was used, marked activation of kinase activity due to a dominant effect of acetyl-CoA was associated with acetylation of a major portion of sites in the complex and with a small portion undergoing acylation with propionyl-CoA. Those results were rapidly achieved in a manner independent of the order of addition of the two CoA esters. That indicates that tight slowly reversible binding of acetyl-CoA is not involved in kinase activation. High levels of propionyl-CoA greatly reduced acetylation by acetyl-CoA and nearly prevented activation of kinase activity by acetyl-CoA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modification of sterculia gum with methacrylic acid to prepare a novel drug delivery system

The present paper deals with the modification of the sterculia gum with methacrylic acid (MAAc) to hydrogels for use in drug delivery. The hydrogels were characterized by SEMs, FTIR and swelling studies. The release dynamics of model anti-ulcer drug (ranitidine hydrochloride) from the hydrogels has been studied for the evaluation of the release mechanism. The values of the diffusion exponent 'n' (0.55, 0.54 and 0.59) and gel characteristic constant 'k' (2.109 x 10(-2), 3.698 x 10(-2) and 2.427 x 10(-2)) have been obtained, respectively, in distilled water, pH 2.2 buffer and pH 7.4 buffer. The release of the drug from the hydrogels occurred through non-Fickian diffusion mechanism.