Reported here are the competitive 18 O/ 16 O kinetic isotope effects ( 18 O KIEs) on k cat /K m (O 2 ) for three non-heme iron enzymes that activate O 2 at an iron center coordinated by a 2-His-1-carboxylate facial triad: taurine dioxygenase (TauD), S-(2)-hydroxypropylphosphonic acid epoxidase (HppE), and 1-aminocyclopropyl-1-carboxylic acid oxidase (ACCO). The comparison of the measured 18 O KIEs with calculated 18 O equilibrium isotope effects ( 18 O EIEs) reveals an excellent correlation with the proposed mechanisms for these enzymes. 18 O KIEs of 1.0104 ± 0.0002 (TauD), 1.0120 ± 0.0002 (HppE), and 1.0215 ± 0.0005 (ACCO) suggest the formation in the rate limiting step of O 2 activation of an Fe III -alkylperoxo, Fe III -OOH, and Fe IV =O species, respectively. By probing only the steps from initial O 2 binding up to and including the first irreversible step of O 2 activation, the measured 18 O KIEs can be a valuable companion to pre-steady state kinetic analyses in studying the complex catalytic mechanisms of non-heme iron enzymes.The O 2 -activating, non-heme iron enzymes catalyze a wide range of oxygenation and oxidation reactions with important biological implications, such as DNA repair, hypoxic response, collagen biosynthesis, and histone demethylation. 1 Most of these enzymes contain a single iron center coordinated by two His and one Asp/Glu residues in a tridentate binding motif referred to as "2-His-1-carboxylate facial triad". Understanding the O 2 -activation process for these enzymes may provide key insights into the source of their divergent substrate specificity despite similarly coordinated active site metal centers. HppE is a reductase-dependent non-heme iron enzyme that catalyzes the epoxidation of S-HPP, the last step in the biosynthesis of the antibiotic fosfomycin. 9 The mechanism of HppE is not as well known as for TauD, formation of an Fe III -OOH species being proposed to involve either a hydrogen atom transfer (HAT) from S-HPP or proton-coupled electron transfer (PCET) from the reductant. 10 The measured 18 O KIE for HppE is 1.0120 ± 0.0002 at 25 °C, using FMN in the presence of NADH as the reductant (Figure 1

Abstract1-Deoxy-D-xylulose 5-phosphate (DXP) reductoisomerase (DXR, also known as methyl-D-erythritol 4-phosphate (MEP) synthase) is a NADPH-dependent enzyme, which catalyzes the conversion of DXP to MEP in the non-mevalonate pathway of isoprene biosynthesis. Two mechanisms have been proposed for the DXR-catalyzed reaction. In the α-ketol rearrangement mechanism, the reaction begins with deprotonation of the C-3 hydroxyl group followed by a 1,2-migration to give methylerythrose phosphate, which is then reduced to MEP by NADPH. In the retroaldol/aldol rearrangement mechanism, DXR first cleaves the C3-C4 bond of DXP in a retroaldol manner to generate a three-carbon and a two-carbon phosphate bimolecular intermediate. These two species are then reunited by an aldol reaction to form a new C-C bond, yielding an aldehyde intermediate. Subsequent reduction by NADPH affords MEP. To differentiate these mechanisms, we have prepared [3-2 H]-and [4-2 H]-DXP and carried out a competitive secondary kinetic isotope effect (KIE) study of the DXR reaction. The normal 2° KIEs observed for [3-2 H]-and [4-2 H]-DXP provide compelling evidence supporting a retroaldol/aldol mechanism for the rearrangement catalyzed by DXR, with the rate-limiting step being cleavage of the C3-C4 bond of DXP.Terpenoids are a large family of secondary metabolites, consisting of more than 55,000 members, that are widely distributed in nature and rich in biological activities. 1,2 Terpenoids are biosynthesized starting with two 5-carbon isoprene units, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which have long been established to be derived from acetate in a pathway involving mevalonic acid as the key intermediate. 3 However, a new mevalonate-independent isoprene source has recently been discovered in eubacteria, archeabacteria, algae, and in the plastids of plants. [4][5][6] Since this pathway is absent in mammals but is essential for many pathogens, including Plasmodium falciparum 7 and Mycobacterium tuberculosis ,8 all enzymes in this pathway are potential antibacterial targets. 9The first committed step of this non-mevalonate pathway is the conversion of 1-deoxy-Dxylulose 5-phosphate (DXP, 1) to methyl-D-erythritol 4-phosphate (MEP, 2), catalyzed by the NADPH-dependent enzyme, DXP reductoisomerase (DXR, also known as MEP synthase, see Scheme 1). 10 Since MEP is the first metabolite specific to this pathway, this biosynthetic route is commonly referred to as the MEP pathway. Two mechanisms have been proposed for the Email: h.w.liu@mail.utexas.edu . DXR-catalyzed reaction (Scheme 1). In the α-ketol rearrangement mechanism (route A), the reaction begins with deprotonation of the C-3 hydroxyl group followed by a 1,2-(C4-to-C2)-migration to give methylerythrose phosphate (3), which is then reduced to MEP (2) by NADPH. NIH Public AccessIn the retroaldol/aldol mechanism (route B), DXR first cleaves the C3-C4 bond of 1 in a retroaldol manner to generate a three-carbon (4) and a two-carbon phosphate (5) Figure S1). 17 This phenomen...

[reaction: see text] 1-deoxy-D-xylulose-5-phosphate (DXP) reductoisomerase is a NADPH-dependent enzyme catalyzing the conversion of DXP to methyl-D-erythritol 4-phosphate (MEP). In this study, each of the hydroxyl groups in DXP and one of its C-1 hydrogen atoms, were separately replaced with a fluorine atom and the effect of the substitution on the catalytic turnover was examined. It was found that the 1-fluoro-DXP is a poor substrate, while both 3- and 4-fluoro-DXP behave as noncompetitive inhibitors.

Abstract(S)-2-hydroxypropylphosphonic acid epoxidase (HppE) catalyzes the epoxide ring closure of (S)-HPP to form fosfomycin, a clinically useful antibiotic. Early investigation showed that its activity can be reconstituted with Fe(II), FMN, NADH, and O 2 , and identified HppE as a new type of mononuclear non-heme iron-dependent oxygenase involving high valent iron-oxo species in the catalysis. However, a recent study showed that the Zn(II)-reconstituted HppE is active, and HppE exhibits modest affinity for FMN. Thus, a new mechanism is proposed in which the active site bound Fe 2+ or Zn 2+ serves as a Lewis acid to activate the 2-OH group of (S)-HPP, and the epoxide ring is formed by the attack of the 2-OH group at C-1 coupled with the transfer of the C-1 hydrogen as a hydride ion to the bound FMN. To distinguish between these mechanistic discrepancies, we reexamined the bioautography assay, the basis for the alternative mechanism, and showed that Zn(II) cannot replace Fe(II) in the HppE reaction, and NADH is indispensable. Moreover, we demonstrated that the proposed role for FMN as a hydride acceptor is inconsistent with the finding that FMN cannot bind to HppE in the presence of substrate. In addition, using a newly developed HPLC assay we showed that several non-flavin electron mediators could replace FMN in the HppE-catalyzed epoxidation. Taken together, these results argue against the newly proposed "nucleophilic displacement-hydride transfer" mechanism, but are fully consistent with the previously proposed iron-redox mechanism for HppE catalysis, which is unique within the mononuclear non-heme iron enzyme superfamily.Fosfomycin (1) is a clinically useful antibiotic (1) for the treatment of lower urinary tract infections (2) and limb-threatening diabetic foot infections (3). It is also effective against methicillin-resistant (4) and vancomycin-resistant (5) strains of Staphylococcus aureus. The antimicrobial activity of fosfomycin has been attributed to the inactivation of UDP-GlcNAc-3-O-enolpyruvyltransferase (MurA), which catalyzes the first committed step in the biosynthesis of peptidoglycan, the main component of the cell wall (6,7).Fosfomycin is biosynthetically derived from (S)-2-hydroxypropylphosphonic acid (2, (S)-HPP) (8,9). The conversion of (S)-HPP to fosfomycin (1) is catalyzed by HPP epoxidase (HppE) (10,11). A mononuclear non-heme iron in HppE active site is essential for enzyme activity (12). Coordination of the iron by His138, Glu142 and His180, the 2-H-1-D/E facial triad, was first implicated by sequence alignment (13) and site-directed mutagenesis studies (14), and was later confirmed by an X-ray crystal structure (15). Earlier research also showed that molecular oxygen is essential for the reaction (11,12). However, no oxygen atoms from O 2 is incorporated into the fosfomycin product (9,11,12). Instead, the oxygen atom of the epoxy *To whom correspondence should be addressed. Fax: 512-471-2746, E-mail: h.w.liu@mail.utexas NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author M...