NMR-assisted crystallography—the integrated application of solid-state NMR, X-ray crystallography, and first-principles computational chemistry—holds significant promise for mechanistic enzymology: by providing atomic-resolution characterization of stable intermediates in enzyme active sites, including hydrogen atom locations and tautomeric equilibria, NMR crystallography offers insight into both structure and chemical dynamics. Here, this integrated approach is used to characterize the tryptophan synthase α-aminoacrylate intermediate, a defining species for pyridoxal-5′-phosphate–dependent enzymes that catalyze β-elimination and replacement reactions. For this intermediate, NMR-assisted crystallography is able to identify the protonation states of the ionizable sites on the cofactor, substrate, and catalytic side chains as well as the location and orientation of crystallographic waters within the active site. Most notable is the water molecule immediately adjacent to the substrate β-carbon, which serves as a hydrogen bond donor to the ε-amino group of the acid–base catalytic residue βLys87. From this analysis, a detailed three-dimensional picture of structure and reactivity emerges, highlighting the fate of the L-serine hydroxyl leaving group and the reaction pathway back to the preceding transition state. Reaction of the α-aminoacrylate intermediate with benzimidazole, an isostere of the natural substrate indole, shows benzimidazole bound in the active site and poised for, but unable to initiate, the subsequent bond formation step. When modeled into the benzimidazole position, indole is positioned with C3 in contact with the α-aminoacrylate C β and aligned for nucleophilic attack. Here, the chemically detailed, three-dimensional structure from NMR-assisted crystallography is key to understanding why benzimidazole does not react, while indole does.
more »
« less
Evidence for the chemical mechanism of RibB (3,4-dihydroxy-2-butanone 4-phosphate synthase) of riboflavin biosynthesis.
RibB (3,4-dihydroxy-2-butanone 4-phosphate synthase) is a magnesium-dependent enzyme that excis-es the C4 of D-ribulose-5-phosphate (D-Ru5P) as formate. This chemistry forms the four-carbon sub-strate for RibE (lumazine synthase) that is incorporated into the xylene moiety of lumazine and ulti-mately the riboflavin isoalloxazine. The reaction was first identified in studies by Bacher and cowork-ers in the early 1990s and a chemical mechanism hypothesis offered by these researchers has become the consensus mechanism despite minimal direct evidence. In addition, X-ray crystal structures of RibB typically show two metal ions when solved in the presence of non-native metals and/or liganding non-substrate analogues and the concensus hypothetical mechanism has incorporated this cofactor set. We have used a variety of biochemical approaches to further characterize the chemistry catalyzed by RibB from Vibrio cholera (VcRibB). We show that full activity is achieved at metal ion concentra-tions equal to the enzyme concentration indicating that only one metal ion is required for catalysis. This was confirmed from EPR of the enzyme reconstituted with manganese and crystal structures ob-tained from soaking with the native substrate, D-Ru5P. These data definitively show the involvement of a single active site metal ion. The slow rate of turnover of VcRibB was used to identify two transient species prior to the formation of products using acid quench of single turnover reactions in combina-tion with NMR for singly and fully 13C-labelled D-Ru5P. The data indicate that dehydration of C1 forms the first transient species that then undergoes rearrangement by a 1,2 migration that fuses C5 to C3 and renders C4 hydrated as a gem diol that is poised for elimination as formate. Time-dependent Mn2+ soaks of VcRibB-D-Ru5P co-crystals provided structures that show accumulation in crystallo of the same intermediate states as observed with acid-quench and NMR. Collectively these data reveal for the first time crucial transient chemical states in the mechanism of RibB.
more »
« less
- Award ID(s):
- 1904480
- NSF-PAR ID:
- 10337149
- Date Published:
- Journal Name:
- Journal of the American Chemical Society
- Volume:
- in press
- ISSN:
- 1943-2984
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
more » « less
Abstract Experimental observations of enzymes under active turnover conditions have brought new insight into the role of protein motions and allosteric networks in catalysis. Many of these studies characterize enzymes under dynamic chemical equilibrium conditions, in which the enzyme is actively catalyzing both the forward and reverse reactions during data acquisition. We have previously analyzed conformational dynamics and allosteric networks of the alpha subunit of tryptophan synthase under such conditions using NMR. We have proposed that this
working state represents a four to one ratio of the enzyme bound with the indole‐3‐glycerol phosphate substrate (E:IGP) to the enzyme bound with the products indole and glyceraldehyde‐3‐phosphate (E:indole:G3P). Here, we analyze the inactive D60N variant to deconvolute the contributions of the substrate‐ and products‐bound states to theworking state. While the D60N substitution itself induces small structural and dynamic changes, the D60N E:IGP and E:indole:G3P states cannot entirely account for the conformational dynamics and allosteric networks present in theworking state. The act of chemical bond breakage and/or formation, or possibly the generation of an intermediate, may alter the structure and dynamics present in theworking state. As the enzyme transitions from the substrate‐bound to the products‐bound state, millisecond conformational exchange processes are quenched and new allosteric connections are made between the alpha active site and the surface which interfaces with the beta subunit. The structural ordering of the enzyme and these new allosteric connections may be important in coordinating the channeling of the indole product into the beta subunit. -
Dihydropyrimidine dehydrogenase (DPD) catalyzes the initial step in the catabolism of the pyrimidines uracil and thymine. Crystal structures have revealed an elaborate subunit architecture consisting of two flavin cofactors, apparently linked by four Fe4S4 centers. Analysis of the DPD reaction(s) equilibrium position under anaerobic conditions revealed a reaction that favors dihydropyrimidine formation. Single-turnover analysis shows biphasic kinetics. The serine variant of the candidate general acid, cysteine 671, provided enhanced kinetic resolution for these phases. In the first event, one subunit of the DPD dimer takes up two electrons from NADPH in a reductive activation step. Spectrophotometric deconvolution suggests that thes electrons reside on one of the two flavins. That oxidation of the enzyme by dioxygen can be suppressed by the addition of pyrimidine, is consistent with these electrons residing on the FMN. The second phase involves further oxidation of NADPH and concomitant reduction of the pyrimidine substrate. During this phase no net reduction of DPD cofactors is observed indicating that the entire cofactor set acts as a wire, transmitting electrons from NADPH to the pyrimidine rapidly. This indicates that the availability of the proton from C671 general acid controls the transmittance of electrons from NADPH to the pyrimidine. Acid quench and HPLC product analysis of single-turnover reactions with limiting NADPH confirmed 2:1, NADPH:pyrimidine stoichiometry for the enzyme accounting for successive activation and pyrimidine reduction. These data support an alternating subunit model in which one protomer is activated and turns over before the other subunit can be activated and enter catalysis.more » « less
-
more » « less
Radiation of the plant pyridoxal 5′-phosphate (PLP)-dependent aromatic
l -amino acid decarboxylase (AAAD) family has yielded an array of paralogous enzymes exhibiting divergent substrate preferences and catalytic mechanisms. Plant AAADs catalyze either the decarboxylation or decarboxylation-dependent oxidative deamination of aromaticl -amino acids to produce aromatic monoamines or aromatic acetaldehydes, respectively. These compounds serve as key precursors for the biosynthesis of several important classes of plant natural products, including indole alkaloids, benzylisoquinoline alkaloids, hydroxycinnamic acid amides, phenylacetaldehyde-derived floral volatiles, and tyrosol derivatives. Here, we present the crystal structures of four functionally distinct plant AAAD paralogs. Through structural and functional analyses, we identify variable structural features of the substrate-binding pocket that underlie the divergent evolution of substrate selectivity toward indole, phenyl, or hydroxyphenyl amino acids in plant AAADs. Moreover, we describe two mechanistic classes of independently arising mutations in AAAD paralogs leading to the convergent evolution of the derived aldehyde synthase activity. Applying knowledge learned from this study, we successfully engineered a shortened benzylisoquinoline alkaloid pathway to produce (S)-norcoclaurine in yeast. This work highlights the pliability of the AAAD fold that allows change of substrate selectivity and access to alternative catalytic mechanisms with only a few mutations. -
more » « less
Two amino acid variants in soybean serine hydroxymethyltransferase 8 (SHMT8) are associated with resistance to the soybean cyst nematode (SCN), a devastating agricultural pathogen with worldwide economic impacts on soybean production. SHMT8 is a cytoplasmic enzyme that catalyzes the pyridoxal 5‐phosphate‐dependent conversion of serine and tetrahydrofolate (THF) to glycine and 5,10‐methylenetetrahydrofolate. A previous study of the P130R/N358Y double variant of SHMT8, identified in the SCN‐resistant soybean cultivar (cv.) Forrest, showed profound impairment of folate binding affinity and reduced THF‐dependent enzyme activity, relative to the highly active SHMT8 in cv. Essex, which is susceptible to SCN. Given the importance of SCN‐resistance in soybean agriculture, we report here the biochemical and structural characterization of the P130R and N358Y single variants to elucidate their individual effects on soybean SHMT8. We find that both single variants have reduced THF‐dependent catalytic activity relative to Essex SHMT8 (10‐ to 50‐fold decrease in
k cat/K m) but are significantly more active than the P130R/N368Y double variant. The kinetic data also show that the single variants lack THF‐substrate inhibition as found in Essex SHMT8, an observation with implications for regulation of the folate cycle. Five crystal structures of the P130R and N358Y variants in complex with various ligands (resolutions from 1.49 to 2.30 Å) reveal distinct structural impacts of the mutations and provide new insights into allosterism. Our results support the notion that the P130R/N358Y double variant in Forrest SHMT8 produces unique and unexpected effects on the enzyme, which cannot be easily predicted from the behavior of the individual variants.
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- The DOI is not currently available.
