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Characterization of 1,2-propanediol dehydratases reveals distinct mechanisms for B12-dependent and glycyl radical enzymes Benjamin Levin, and Emily P Balskus Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00164 • Publication Date (Web): 12 Mar 2018 Downloaded from http://pubs.acs.org on March 12, 2018
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Biochemistry
Characterization of 1,2-propanediol dehydratases reveals distinct mechanisms for B12-dependent and glycyl radical enzymes Benjamin J. Levin and Emily P. Balskus* Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138 United States
Supporting Information Placeholder ABSTRACT: Propanediol dehydratase (PD), a recently characterized member of the glycyl radical enzyme (GRE) family, uses protein-based radicals to catalyze the chemically challenging dehydration of (S)-1,2-propanediol. This transformation is also performed by the well-studied enzyme B12-dependent propanediol dehydratase (B12-PD) using an adenosylcobalamin cofactor. Despite the prominence of PD in anaerobic microorganisms, it remains unclear if the mechanism of this enzyme is similar to that of B12-PD. Here we report oxygen-18 labeling experiments that suggest PD and B12-PD employ distinct mechanisms. Unlike B12PD, PD appears to catalyze the direct elimination of a hydroxyl group from an initially formed substrate-based radical, avoiding the generation of a 1,1-gem diol intermediate. Our studies provide further insights into how GREs perform elimination chemistry and highlight how nature has evolved diverse strategies for catalyzing challenging reactions.
Enzymes use protein- and cofactor-based radicals to perform challenging transformations that cannot be achieved with twoelectron chemistry.1 Glycyl radical enzymes (GREs) are a large family of proteins found in anaerobic microbes that promote a diverse set of reactions by employing a glycine-centered radical intermediate (Figure 1A).2 This key protein-based radical is installed posttranslationally by GRE activating enzymes (AEs), members of the radical S-adenosylmethionine (SAM) enzyme superfamily.3 These enzymes use a 5′-deoxyadenosyl radical (5′dA•) generated from SAM to abstract an α-hydrogen from a conserved active site glycine in their partner GREs. The glycyl radical abstracts a hydrogen atom from a conserved cysteine, and the resulting thiyl radical reacts with bound substrate. Examples of reactions performed by GREs include C–C bond formation (benzylsuccinate synthase), C–C bond cleavage (pyruvate formatelyase, p-hydroxyphenylacetate decarboxylase), C–N bond cleavage (choline trimethylamine-lyase), ribonucleotide reduction (Class III ribonucleotide reductase (RNR)), and dehydration (glycerol dehydratase). GREs have been identified in high abundance and exceptional diversity in the human gut microbiome.4 One particularly important GRE-mediated host-gut microbe interaction is the metabolism of L-fucose derived from host glycans (Figure 1B).5–7 Human gut bacteria use the L-fucose utilization (fuc) pathway to yield dihydroxyacetone phosphate and (S)-1,2-propanediol.8 Organisms possessing propanediol utilization (pdu) genes can further convert (S)-1,2-propanediol to propionaldehyde, which can be either reduced to 1-propanol or oxidized to produce the beneficial shortchain fatty acid propionate.9–11
Figure 1. Overview of glycyl radical enzymes (GREs) and Lfucose metabolism. (A) General mechanism of GREs. (B) The role of (S)-1,2-propanediol in L-fucose utilization. The dehydration of (S)-1,2-propanediol to propionaldehyde is chemically challenging, as the C2-hydroxyl group is a poor leaving group and the C1-hydrogen atoms are not acidic. Enzymes catalyzing the dehydration of 1,2-diols have therefore evolved to use radical chemistry.12 Adenosylcobalamin-dependent enzymes that dehydrate 1,2-propanediol and glycerol were discovered by the Abeles laboratory in the 1960s, and seminal isotope labeling experiments from the Abeles and Arigoni groups established that B12-dependent propanediol dehydratase (B12-PD) mediates an intramolecular rearrangement.13,14 From these and subsequent studies, a consensus mechanism has emerged for this enzyme (Scheme 1).12 The binding of substrate to B12-PD initiates homolysis of the adenosylcobalamin cofactor Co–C bond, and the resulting 5′-dA• abstracts a hydrogen atom from C1 of the substrate,
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generating an α-hydroxyalkyl radical. This intermediate then rearranges to the 1,1-dihydroxyprop-2-yl radical via a stereospecific hydroxyl group migration from C2 to C1 facilitated by partial protonation and deprotonation of the migrating and spectator hydroxyl groups, respectively, and by a Ca2+ ion that interacts with both hydroxyl groups. The resulting C2-centered radical abstracts a hydrogen atom from 5′-deoxyadenosine (5′-dA), regenerating the cofactor-based radical, and the 1,1-gem diol intermediate collapses stereoselectively in the B12-PD active site to yield propionaldehyde.
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mations and reveal mechanistic differences between GREs and B12-dependent enzymes that have evolved identical functions. Hydroxyl migration
A H• abstraction
OH
OH
Me
Me
H2O
OH
Me
OH
OH
Differentially 18 O-labeled
H2O
H
O
Me
Scheme 1. Consensus mechanism for B12-PD
O
Me
OH
18
O label?
H Direct elimination
B O Me
0.45 equiv H 218O, rt 0.002 equiv (R,R)- or (S,S)(salen)Co III•OAc
18
O
Me
0.45 equiv H 2O, rt 0.002 equiv (R,R)- or (S,S)(salen)Co III•OAc
OH Me
18OH
Me
35%, 99% ee, 96% Oxygen-18 18OH
Me
OH
43%, 98% ee, 96% Oxygen-18
OH
or
18OH
36%, 99% ee, 96% Oxygen-18
or
18OH
Me
OH
39%, 99% ee, 94% Oxygen-18
Figure 2. Design of 18O-labeling experiments to probe the mechanism of PD. (A) Two potential mechanisms for radical enzymemediated diol dehydration. (B) Asymmetric synthesis of differentially 18O-labeled 1,2-propanediol substrates.
More recently, pdu gene clusters were identified that lacked an encoded B12-PD but encoded a putative GRE,15 which was later confirmed to be a B12-independent 1,2-propanediol dehydratase (PD).4,16 Like other predicted GRE dehydratases, PD is highly abundant and widely distributed in the gut microbiomes of healthy humans, suggesting PD and L-fucose metabolism play a crucial role in the host-gut microbe relationship.4 Despite this prominence, it remains unclear how PD and other GREs catalyze dehydration. The highly similar GRE glycerol dehydratase (GD) was initially proposed to operate analogously to the B12-dependent dehydratases, with a thiyl radical intermediate abstracting glycerol’s C1-hydrogen atom while glutamate and histidine residues facilitate hydroxyl migration.17 However, a QM/MM study cast doubt on this proposal, revealing that shifting the C2-hydroxyl group closer to C1 has a barrier of > 20 kcal/mol versus < 6 kcal/mol for eliminating the C2-hydroxyl group from the initial αhydroxyalkyl substrate-based radical.18 This direct elimination resembles the first half of the proposed mechanism for the GRE Class III RNR.19 Here, we use oxygen-18 labeling experiments to provide an initial mechanistic comparison between PD and B12-PD. Though both enzymes likely form the same initial substrate-based radical, assays with labeled substrates provide no evidence that PD catalyzes a 1,2-hydroxyl migration, supporting the direct elimination proposal. Results with B12-PD corroborate the involvement of hydroxyl migration. These experiments clarify how PD and other GREs important to human health carry out challenging transfor-
We began by biochemical characterizing PD and its activating enzyme (PD-AE) to verify they form a canonical GRE/activase pair. As described previously, we heterologously expressed the PD and PD-AE from Roseburia inulinivorans A2-194 in Escherichia coli as His6-tagged fusions.4 PD-AE possessed the CX3CX2C motif characteristic of radical SAM enzymes,3 and both its UV-visible spectra in the presence and absence of an external reductant (Figure S2) as well the presence of iron and sulfur (2.80±0.03 equiv of iron and 2.71±0.02 equiv of sulfur per monomer) were consistent with an [4Fe–4S] cluster. The levels of incorporated iron and sulfide suggest our enzyme preparation contains a mixture of properly reconstituted and inactive [4Fe–4S] clusters. We verified PD-AE converted S-adenosylmethionine (SAM) into 5ʹ-dA in the presence of 5-deazariboflavin and dithiothreitol using high performance liquid chromatography (HPLC) (Figure S3) and liquid chromatography-mass spectrometry (LCMS) (Figure S4) as is typical for radical SAM enzymes, supporting PD-AE’s classification as a canonical GRE-AE. Next, we used stable-isotope labeling experiments to study the mechanisms of PD and B12-PD. Experiments with differentially labeled diol substrates have not been performed with dehydrating GREs. To explore the possibility of a hydroxyl migration in the PD-catalyzed reaction, we used the Jacobsen hydrolytic kinetic resolution to synthesize all four possible 18O-labeled 1,2propanediol stereoisomers in high enantiomeric excess (Figure 2).20 Although similar substrates have been constructed previously, our oxygen-18 labeled 1,2-propanediols are labeled to a much greater extent (>95% vs. ~10%).14 In addition, previous studies did not test enantioenriched 1,2-(2-18O)propanediols with PD or B12-PD. We incubated the four isotopomers of 1,2(18O)propanediol individually with activated PD and yeast alcohol dehydrogenase, and we used gas chromatography-mass spectrometry (GC-MS/MS) to assess the presence of oxygen-18 in the 1propanol product (Table 1 and Figure S5). We observed nearly complete retention of the label with both enantiomers of 1,2-(118 O)propanediol and nearly complete loss of the label with both enantiomers of 1,2-(2-18O)propanediol. These results are distinct from those observed by Arigoni and co-workers and do not afford analogous support for a mechanism involving hydroxyl migration. Instead, they suggest GRE-mediated dehydration reactions pro-
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Biochemistry
ceed in a different manner from those performed by B12dependent enzymes.
Table 1. Dehydration of 18O-labeled substratesa
Oxygen-18 Enrichment (%) Substrate
1-Propanol 1,2-Propanediol PD
B12-PD
K. oxytoca extract
96.1±0.4
91.4±1.1
51.7±0.1
61±2
96.1±0.5
92.2±0.5
49.6±1.4
13.8±0.2
96±3
2.5±0.4
47.7±0.3
14.2±0.3
94±5
2.5±0.4
48.9±0.2
48±5
values of α-hydroxylalkyl radicals are ~5 pH units more acidic than the corresponding alcohols,23 and E440 is well positioned to perform this chemistry. The electron density from this ketyl radical fills the σ* of the C2–O bond, triggering loss of the hydroxyl group, which is likely protonated by H166. The direct elimination of a hydroxyl group adjacent to an α-hydroxyalkyl radical has precedence in enzymatic catalysis as all known types of RNR demonstrate this reactivity. To complete the reaction, the propanal-2-yl radical abstracts a hydrogen atom from C817, regenerating the protein-based radical intermediates.
Scheme 2. Proposed mechanism for PD
a 1,2-Propanediol (10 mM), PD or B12-PD (1 µM), yeast alcohol dehydrogenase (35 µM), NADH (10 mM), adenosylcobalamin (B12-PD only) (15 µM), Tris•HCl (25 mM, pH=7.5), NaCl (50 mM), 1.5 h, 23 °C (PD), 37 °C (B12-PD). 18O-Enrichment measured by GC-MS. Mean±SD are from three replicates.
We also tested the reactivity of B12-PD toward these isotopelabeled substrates to more directly compare the evidence for the two mechanistic proposals (Table 1). We heterologously expressed B12-PD from Klebsiella oxytoca ATCC 8724 in E. coli using a previously reported procedure (Figure S1).21 After verifying the enzyme’s activity, we incubated B12-PD and yeast alcohol dehydrogenase with the oxygen-18 labeled substrates. Regardless of which 1,2-(18O)propanediol was used, the product 1-propanol always contained ~50% oxygen-18 (Table 1 and Figure S6). This scrambling of oxygen atoms supports the existence of the 1,1-gem diol intermediate. Surprisingly, the results we obtained with purified B12-PD were inconsistent with the original oxygen-18 labeling experiments performed by Arigoni et al. who used cell-free extracts of K. oxytoca ATCC 8724 instead of purified B12-PD and less extensively labeled substrates. To explain this inconsistency, we replicated their work, using the same strain of K. oxytoca and generating cell-free extracts according to an Abeles group protocol.22 When the cell-free extracts, NADH, and alcohol dehydrogenase were incubated with (S)-1,2-(1-18O)propanediol and (R)-1,2-(118 O)propanediol, we observed 61% and 14% retention of the oxygen-18 label, respectively (Table 1 and Figure S7). Complementary results were observed with both enantiomers of 1,2-(218 O)propanediol, confirming that the fate of each oxygen atom depends on the substrate stereochemistry. These experiments are consistent with the Arigoni group’s results, yet it remains unclear which component(s) of the cell-free extract alter the stereochemical outcome of this reaction. Regardless, our experiments with purified B12-PD and with K. oxytoca lysate both confirm B12-PD mediates hydroxyl group migration. The results of these experiments allow us to propose a mechanism for PD (Scheme 2). Analogously to other GREs, the glycine centered radical, derived from G817, abstracts a hydrogen atom from C438, generating a thiyl radical that in turn abstracts a hydrogen atom from C1 of the 1,2-propanediol substrate. Results from our oxygen-18 experiments and previous calculations with GD suggest that PD catalyzes the direct elimination of the C2hydroxyl group from this substrate-based radical.18 This step is likely triggered by deprotonating the C1-hydroxyl group. The pKa
Structural data also corroborates this proposed mechanism for PD. The crystal structure of PD with (S)-1,2-propanediol bound reveals how C438 can interact with G817 and the pro-S C1hydrogen atom of the substrate, supporting its role in reaction initiation (Figure S8).16 The dihedral angle between the two hydroxyl groups of (S)-1,2-propanediol in the crystal structure is 57.4°, and we can infer that the pro-S hydrogen atom of C1 is antiperiplanar to the C2-hydroxyl group, facilitating dehydration. Furthermore, E440 and H166 are well-positioned to deprotonate and protonate the C1 and C2-hydroxyl groups, respectively. These residues are essential for PD activity and conserved in all charcterized GRE dehydratases.4 The GRE deaminase choline trimethylamine-lyase binds choline similarly to how PD binds (S)1,2-propanediol,24 suggesting GRE dehydratases and 1,2eliminases use similar mechanisms. Though our labeling experiment does not completely rule out the possibility of migration followed by stereospecific dehydration of a 1,1-gem diol intermediate, these studies together with computational and structural studies provide strong support for a direct elimination mechanism. The wide distribution of GREs and their roles in important microbial metabolic processes motivate our desire to understand their mechanisms. For PD and B12-PD, chemical differences between their cofactors could explain why they evolved distinct mechanisms to catalyze the same transformation. To initiate catalysis, B12-PD uses the highly reactive 5′-dA• (5′-dA–H bond dissocation energy (BDE), 99.8 kcal/mol)25 to abstract the C1hydrogen (C–H BDE, 92 kcal/mol)26. However, the thiyl radical in PD is less reactive than 5′-dA• (S–H BDE, ~87 kcal/mol)27, but hydrogen bonding between E440 and the C1-hydroxyl group may decrease the C1–H BDE.26 Similarly, the propanal-2-yl radical intermediate formed by PD would not be reactive enough (C–H BDE, ~90 kcal/mol)28 to abstract a hydrogen atom from 5′-dA to regenerate the 5′-dA• cofactor. Conversely, the product-centered radical generated by B12-PD cannot be delocalized and is therefore reactive enough (C–H BDE, ~98.6 kcal/mol)27 to regenerate the adenosylcobalamin cofactor. In addition, while the Ca2+ ion in B12-PD lowers the barrier to hydroxyl migration, no such Lewis
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acid is found in PD, further suggesting key mechanistic differences. Two other characterized GREs, choline-trimethylamine lyase and RNR (Class III), have B12-dependent counterparts (ethanolamine-ammonia lyase and RNR (Class II)), hinting that these protein families may have additional functional overlap. Future efforts to understand the functions of both enzyme families will continue to enhance our understanding of host-microbe interactions as they pertain to carbohydrate metabolism.
ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Experimental methods, synthetic details, and additional figures (PDF)
AUTHOR INFORMATION Corresponding Author *
[email protected] ORCID Benjamin J. Levin: 0000-0003-3220-9303 Emily P. Balskus: 0000-0001-5985-5714
Notes The authors declare no competing financial interests.
ACKNOWLEDGMENT Financial support was provided by the Packard Fellowship for Science and Engineering and the First 100 W Program established by the TomKat Foundation (E.P.B.). B.J.L. acknowledges support from the National Science Foundation Graduate Research Fellowship Program (DGE1144152). We thank Prof. Takamasa Tobimatsu (Okayama University) for providing the plasmid pUCDD11 and Jennifer X. Wang (Harvard University) for assistance with MS experiments.
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(8) Chen, Y. M., Zhu, Y., and Lin, E. C. (1987) The organization of the fuc regulon specifying L-fucose dissimilation in Escherichia coli K12 as determined by gene cloning, Mol. Gen. Genet. 210, 331–337. (9) Bobik, T. A., Havemann, G. D., Busch, R. J., Williams, D. S., and Aldrich, H. C. (1999) The propanediol utilization (pdu) operon of Salmonella enterica serovar typhimurium LT2 includes genes necessary for formation of polyhedral organelles involved in Coenzyme B12-dependent 1,2-propanediol degradation, J. Bacteriol. 181, 5967–5975. (10) Reichardt, N., Duncan, S. H., Young, P., Belenguer, A., Leitch, C. M., Scott, K. P., Flint, H. J., and Louis, P. (2014) Phylogenetic distribution of three pathways for propionate production within the human gut microbiota, ISME J. 8, 1323–1335. (11) Hosseini, E., Grootaert, C., Verstraete, W., and Van de Wiele, T. (2011) Propionate as a health-promoting microbial metabolite in the human gut, Nutr. Rev. 69, 245–258. (12) Toraya, T. (2014) Cobalamin-dependent dehydratases and a deaminase: Radical catalysis and reactivating chaperones, Arch. Biochem. Biophys. 544, 40–57. (13) Zagalak, B., Frey, P. A., Karabatsos, G. L., and Abeles, R. H. (1966) The stereochemistry of the conversion of D and L 1,2-propanediols to propionaldehyde, J. Biol. Chem. 241, 3028–3035. (14) Rétey, J., Umani-Ronchi, A., Seibl, J., and Arigoni, D. (1966) Zum mechanismus der propandioldehydrase-reaktion, Experientia 22, 502–503. (15) Scott, K. P., Martin, J. C., Campbell, G., Mayer, C. D., and Flint, H. J. (2006) Whole-genome transcription profiling reveals genes upregulated by growth on fucose in the human gut bacterium “Roseburia inulinivorans”, J. Bacteriol. 188, 4340–4349. (16) LaMattina, J. W., Keul, N. D., Reitzer, P., Kapoor, S., Galzerani, F., Koch, D. J., Gouvea, I. E., and Lanzilotta, W. N. (2016) 1,2Propanediol dehydration in Roseburia inulinivorans: Structural basis for substrate and enantiomer selectivity, J. Biol. Chem. 291, 15515–15526. (17) O’Brien, J. R., Raynaud, C., Croux, C., Girbal, L., Soucaille, P., and Lanzilotta, W. N. (2004) Insight into the mechanism of the B12independent glycerol dehydratase from Clostridium butyricum: Preliminary biochemical and structural characterization, Biochemistry 43, 4635– 4645. (18) Feliks, M., and Ullman, G. M. (2012) Glycerol dehydratation by the B12-independent enzyme may not involve the migration of a hydroxyl group: A computational study, J. Phys. Chem. B 116, 7076–7087. (19) Yei, Y., Funk, M. A., Rosado, L. A., Baek, J., Drennan, C. L., and Stubbe, J. (2014) The class III ribonucleotide reductase from Neisseria bacilliformis can utilize thioredoxin as a reductant, Proc. Natl. Acad. Sci. USA 111, E3756–E3765. (20) Schaus, S. E., Brandes, B. D., Larrow, J. F., Tokunaga, M., Hansen, K. B., Gould, A. E., Furrow, M. E., and Jacobsen, E. N. (2002) Highly selective hydrolytic kinetic resolution of terminal epoxides catalyzed by chiral (salen)CoIII complexes. Practical synthesis of enantioenriched terminal epoxides and 1,2-diols, J. Am. Chem. Soc. 124, 1307–1315. (21) Tobimatsu, T., Sakai, T., Hashida, Y., Mizoguchi, N., Miyoshi, S., and Toraya, T. (1997) Heterologous expression, purification, and properties of diol dehydratase, an adenosylcobalamin-dependent enzyme of Klebsiella oxytoca, Arch. Biochem. Biophys. 347, 132–140. (22) Abeles, R. H., and Lee, H. A., Jr. (1961) An intramolecular oxidation-reduction requiring a cobamide coenzyme, J. Biol. Chem. 236, 2347– 2350. (23) Hayon, E. and Simic, M. (1974) Acid–base properties of free radicals in solution, Acc. Chem. Res. 7, 114–121. (24) Bodea, S., Funk, M. A., Balskus, E. P., and Drennan, C. L. (2016) Molecular basis of C–N bond cleavage by the glycyl radical enzyme choline trimethylamine-lyase, Cell Chem. Biol. 23, 1206–1216. (25) Wetmore, S. D., Smith, D. M., Golding, B. T., and Radom, L. (2001) Interconversion of (S)-glutamate and (2S,3S)-3-methylaspartate: A distinctive B12-dependent carbon-skeleton rearrangement, J. Am. Chem. Soc. 123, 7963–7972. (26) Jeffrey, J. L., Terrett, J. A., and MacMillan D. W. C. (2015) O–H hydrogen bonding promotes H-atom transfer from α C–H bonds for Calkylation of alcohols, Science 349, 1532–1536. (27) Blanksby, S. J. and Ellison, G. B. (2003) Bond dissociation energies of organic molecules, Acc. Chem. Res. 36, 255–263. (28) da Silva, G. and Bozzelli, J. W. (2006) Enthalpies of formation, bond dissociation energies, and molecular structures of the n-aldehydes (acetaldehyde, propanal, butanal, pentanal, hexanal, and heptanal) and their radicals, J. Phys. Chem. A 110, 13058–13067.
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Biochemistry
Ade O OH
O
Me H
H N
OH Co
3+
OH
L
+
Me O
Me H
– H2O/H2O B12-dependent enzyme
HN O
1,2-propanediol
O
Me
OH
H – H2O Glycyl radical enzyme
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HN Biochemistry Conserved Page 6 of 10 H N
SAM + e–
O
Gly in GRE
GRE-AE 1 L-Met + 5'-dA 2 Product Substrate HN 3 H HN HN N H H 4 O N N O O 5 H H HS 6 S S Product Substrate 7 8 Radical reaction or HN HN rearrangement 9 H H N N 10 O O H H 11 HS HS 12 Product Substrate 13 B -dependent dehydratase B14 OH Me C–O migration 15 n-Propanol Fuc 16 OH O OH pathway O Me Me OH + H2O 17 OH Me H OH OH 18L-Fucose (S)-1,2-Propanediol Propionaldehyde 19 O ACS Paragon Plus Environment Me Mechanism? 20 O Dehydrating GRE Propionate 21 12
A Page 7 of 10 H• abstraction
OH Me 1
2 3 4 B 5 6Me 7 8 9 10 Me 11 12
Hydroxyl migration
Biochemistry
OH
Me
H2O
OH
Me
OH
OH
Differentially 18 O-labeled
H2O
O
Me
OH
H
O
Me
18
O label?
H Direct elimination
O
0.45 equiv H218O, rt 0.002 equiv (R,R)- or (S,S)(salen)CoIII•OAc
18O
0.45 equiv H2O, rt
OH
18
OH
Me
35%, 99% ee, 96% Oxygen-18 18
OH
or ACS Paragon Plus Environment OH 0.002 equiv (R,R)- or (S,S)(salen)CoIII•OAc
Me
43%, 98% ee, 96% Oxygen-18
OH
or
18
OH
Me
36%, 99% ee, 96% Oxygen-18 18
OH
Me
OH
39%, 99% ee, 94% Oxygen-18
Ado CH2
Biochemistry 3+
O
Me
Co
H
OH
Page 8 of 10 OH
Me
L
1N 2 H143 3 4 5 6 7 8 9N 10 H143 11 12 13 14 15 16 17 18 19 20
O
NH
Ca2+ H
Me H Ado CH2
N O
O
Me
E170
O
H NH
H143
Ca2+ H O O
H Ado CH2
O
Co2+
Co2+
L
L
E170
H 2O H NH
O
Me
Ca2+ H O O
H Ado CH2
N
O
Me
E170
O
H NH
Ca2+ H O O
H143 Ado CH3
Co2+
Co2+
L
L N
H NH
O
Me H143
Ca2+ H O O H
O
E170
ACS Paragon Plus Environment Ado C H2
Co2+ L
O
E170
H
H166
N Page 9 of E440 10 H
N+ OH
H O
O
O
H
H N
H166
N Biochemistry E440 H
N+ OH
H O
O
O
H
H166
N+
Me Me 1 C438 C438 HS 2 H S 3 (! )-1,2-Propanediol C438 SH 4 Propionaldehyde + H2O G817• 5 H H166 H H166 H N N 6N E440 E440 N+ 7N N H HO HO OH O O 8H2O H2 O ACS Paragon Plus Environment O Me C438 C438 O Me 9 Me H S H 10 H S G817–C –H 11 Me
HO
OH O
E440 O C438
HS
H166
HO O HS
E440 O C438
Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 10 of 10
Ade O OH
O
Me H
H N
OH Co3+
OH
L
+
Me O
Me H
– H2O/H2O B12-dependent enzyme
HN O
1,2-propanediol
O
Me
OH
H – H 2O Glycyl radical enzyme
ACS Paragon Plus Environment
