Perry A. Frey

The Radical SAM Superfamily

ABSTRACT The radical S-adenosylmethionine (SAM) superfamily currently comprises more than 2800 proteins with the amino acid sequence motif CxxxCxxC unaccompanied by a fourth conserved cysteine. The charcteristic three-cysteine motif nucleates a [4Fe–4S] cluster, which binds SAM as a ligand to the unique Fe not ligated to a cysteine residue. The members participate in more than 40 distinct biochemical transformations, and most members have not been biochemically characterized. A handful of the members of this superfamily have been purified and at least partially characterized. Significant mechanistic and structural information is available for lysine 2,3-aminomutase, pyruvate formate-lyase, coproporphyrinogen III oxidase, and MoaA required for molybdopterin biosynthesis. Biochemical information is available for spore photoproduct lyase, anaerobic ribonucleotide reductase activation subunit, lipoyl synthase, and MiaB involved in methylthiolation of isopentenyladenine-37 in tRNA. The radical SAM enzymes biochemically characterized to date have in common the cleavage of the [4Fe–4S]1 + –SAM complex to [4Fe–4S]2 +–Met and the 5′ -deoxyadenosyl radical, which abstracts a hydrogen atom from the substrate to initiate a radical mechanism.

The Low Barrier Hydrogen Bond in Enzymatic Catalysis

The proposal that low barrier (i.e. short, very strong) hydrogen bonds (LBHBs) play a role in enzymatic catalysis was first put forth in 1993 and 1994 (1–4). The proposal was accepted by some but rejected by others (5–8). Initial rejection on theoretical grounds has been followed by increasing experimental support, and recent improvements in theory have been able to account for the experimental observations of LBHBs in enzymes (9–15). In this minireview we will explain the original proposal, summarize the experimental data from the past few years, and argue that LBHBs do play important roles in enzymatic reactions.

Understanding enzymic catalysis: the importance of short, strong hydrogen bonds

We have proposed previously that short, strong hydrogen bonds exist in enzyme active sites and that they are important in explaining enzymic rate enhancements. Here, we defend this proposal and provide evidence for likely changes of hydrogen bond strengths during enzymic catalysis.

Radical mechanisms of S-adenosylmethionine-dependent enzymes.

Publisher Summary This chapter discusses the family of S-adenosylmethione (SAM)-dependent enzymes that make use of the 5’-deoxyadenosyl radical, with special reference to the mechanism by which SAM is cleaved reversibly at the active site. One member of the family, lysine 2,3-aminomutase, makes use of the 5’-deoxyadenosyl radical to initiate the molecular rearrangement of a substrate, and the radical is subsequently regenerated in the catalytic cycle, such that SAM functions in a catalytic role as a true coenzyme. The other family members use SAM as a substrate, and the 5’-deoxyadenosyl free radical appears to be an intermediate in an irreversible hydrogen abstraction reaction. The latter enzymes include the pyruvate formate-lyase activating enzyme, the anaerobic ribonucleotide reductase from escherichia coli , biotin synthase, and lipoic acid synthase. The chapter also considers the functions of the 5’-deoxyadenosyl radical and the mechanisms of these diverse reactions. Photolytic or thermal cleavage of adenosylcobalamin produces cob(II)alamin and the 5’- deoxyadenosyl radical through homolytic scission of the Co-C bond. Spectral and kinetic evidence indicate that this same cleavage is brought about at the active sites of adenosylcobalamin-dependent enzymes, and the ensuing 5’-deoxyadenosyl radical is thought to initiate the free radical-based reaction mechanisms catalyzed by those enzymes.

Cloning, Sequencing, Heterologous Expression, Purification, and Characterization of Adenosylcobalamin-dependentd-Lysine 5,6-Aminomutase from Clostridium sticklandii

d-Lysine 5,6-aminomutase fromClostridium sticklandii catalyzes the 1,2-shift of the ε-amino group of d-lysine and reverse migration of C5(H). The two genes encoding 5,6-aminomutase have been cloned, sequenced, and expressed in Escherchia coli. They are adjacent on theClostridial chromosome and encode polypeptides of 57.3 and 29.2 kilodaltons. The predicted amino acid sequence includes a conserved base-off 5′-deoxyadenosylcobalamin binding motif and a 3-cysteine cluster in the small subunit, as well as a P-loop sequence in the large subunit. Activity of the recombinant enzyme exceeds that of the 5,6-aminomutase purified from C. sticklandii by 6-fold, presumably due to the absence of bound, inactive corrinoids in the recombinant enzyme. The K m values for adenosylcobalamin and pyridoxal 5′-phosphate are 6.6 and 1.0 μm, respectively. ATP does not have a regulatory effect on the recombinant protein. The rapid turnover associated inactivation reported for the enzyme purified from Clostridium is also seen with the recombinant form. Aminomutase activity does not depend on structural or catalytic metal ions. Electron paramagnetic resonance experiments with [15N-dimethylbenz-imidazole]adenosylcobalamin demonstrate base-off binding, consistent with other B12-dependent enzymes that break unactivated C—H bonds.

Radicals in enzymatic reactions

Spectroscopic and kinetic evidence for substrate-based radicals in the reactions of lysine 2,3-aminomutase and methane monooxygenase has recently been gathered. Evidence for a protein-based thiyl radical in the mechanism of the action of ribonucleotide reductase has been correlated with the proposed mechanism involving substrate-based radicals. Controversies have arisen about the mechanisms of ribonucleotide reductase and methane monooxygenase reactions.

[2] Galactose-1-phosphate uridylyltransferase: Detection, isolation, and characterization of the uridylyl enzyme

Publisher Summary This chapter discusses the isolation and characterization of the uridylyl enzyme, galactose-1-phosphate uridylyltransferase. Galactose-1-P uridylyltransferase can be purified from Escherichia coli, human erythrocytes, and Saccharomyces cerevisiae. Of these, the enzyme from E. coli is the most thoroughly characterized with respect to molecular properties and mechanism of action. While characterizing the catalytic pathway followed by E. coli galactose-1-P uridylyltransferase, the steady state rates are found to be consistent only with the double displacement (Ping Pong Bi Bi) kinetic model. The intermediate uridylyl-enzyme implied by the model given in the chapter can be isolated in both catalytically active and fully denatured forms. The uridylyl group in this intermediate has been shown to be bonded to N-3 of a histidine residue, and uridylyl group transfer has been shown to proceed with net retention of stereochemical configuration at P α of substrates, which is consistent with a double-displacement mechanism.

[14] Stereochemistry of selected phosphotransferases and nucleotidyltransferases

Publisher Summary In recognition of the experimental imperatives for carrying out stereochemical analyses of chemical reactions, this chapter deals with the methodologies of such research. It describes methods for synthesizing chirally substituted phosphorothioate analogs of biological phosphates. The methods used for assigning configurations to these molecules and the strategies employed in the applications of the foregoing methods to the stereochemical analysis of enzymic substitution at phosphorus are also discussed. The chapter presents the methods by which sulfur may be displaced from chiral phosphorothioates by H2O (or H218O or H217O) to produce chiral phosphates. The availability of chirally substituted phosphorothioates makes them logical precursor molecules for syntheses of chirally substituted phosphates; if stereochemically defined, methods can be devised for displacing sulfur from the chiral centers with 18O or 17O. It is possible to synthesize ATP or ADP with 18O or/and 17O enrichment coupling highly stereoselective or stereospecific enzymatic phosphorylations of ADPβS,β18O or AMPS,18O with the methods discussed in the chapter.

Esherichia coli pyruvate dehydrogenase complex: Improved purification and the flavin content

Abstract The E. coli pyruvate dehydrogenase complex, when purified by published procedures, contains phosphotransacetylase and coenzyme A as trace contaminants as well as one or more spectral contaminants which interfere with spectral and radiochemical experiments. They can be removed by further chromatographic purification on columns of calcium phosphate gel-cellulose. The resulting complexes from E. coli K12 or Crookes strain are indistinguishable with respect to visible spectrum, catalytic activity, and flavin content. The activity is the highest so far reported, 40–42 μmoles DPNH per min per mg of protein, and the flavin content is 1.8–2.4 nanomoles per mg of protein.

Cloning, expression, purification, cofactor requirements, and steady state kinetics of phosphoketolase-2 from Lactobacillus plantarum

The genes xpk1 and xpk2(Delta1-21) encoding phosphoketolase-1 and (Delta1-7)-truncated phosphoketolase-2 have been cloned from Lactobacillus plantarum and expressed in Escherichia coli. Both gene-products display phosphoketolase activity on fructose-6-phosphate in extracts. A N-terminal His-tag construct of xpk2(Delta1-21) was also expressed in E. coli and produced active His-tagged (Delta1-7)-truncated phosphoketolase-2 (hereafter phosphoketolase-2). Phosphoketolase-2 is activated by thiamine pyrophosphate (TPP) and the divalent metal ions Mg(2+), Mn(2+), or Ca(2+). Kinetic analysis and data from the literature indicate the activators are MgTPP, MnTPP, or CaTPP, and these species activate by an ordered equilibrium binding pathway, with Me(2+)TPP binding first and then fructose-6-phosphate. Phosphoketolase-2 accepts either fructose-6-phosphate or xylulose-5-phosphate as substrates, together with inorganic phosphate, to produce acetyl phosphate and either erythrose-4-phosphate or glyceraldehyde-3-phosphate, respectively. Steady state kinetic analysis of acetyl phosphate formation with either substrate indicates a ping pong kinetic mechanism. Product inhibition patterns with erythrose-4-phosphate indicate that an intermediate in the ping pong mechanism is formed irreversibly. Background mechanistic information indicates that this intermediate is 2-acetyl-TPP. The irreversibility of 2-acetyl-TPP formation might explain the overall irreversibility of the reaction of phosphoketolase-2.