Because the sugar moieties of natural products are primarily O-linked, the hydrolytic sensitivity of the glycosidic linkage limits their therapeutic application. One potential solution to this problem is to replace the labile O-glycosidic bond with an enzymatically and chemically stable C-glycosidic bond. In this study, computational analysis of the O-glycosyltransferase LanGT2 and the C-glycosyltransferase UrdGT2 was used to predict the changes necessary to switch the O-glycosylating enzyme to a C-glycosyltransferase. By screening rationally designed LanGT2 mutants a number of LanGT2 variants with C-glycosyltransferase activity were identified. One variant, having 10 amino acid substitutions, revealed the primary region that determines O- versus C-glycosylation. By modeling the active site of this mutant and probing the role of active site residues with alanine substitutions, this work also illuminates the mechanistic features of O- and C-glycosylation.
Keywords
DUF62; halogenation; hydrolase; S-adenosyl-L-methionine; SalinisporaS-Adenosyl-L-methionine (SAM) is a ubiquitous molecule that participates in various biochemical reactions, second only to ATP as the most frequently used enzyme substrate. [1] The recently described SAM-dependent halogenases involved in the biosynthesis of secondary metabolites in actinomycetes represent a new family of SAM-binding proteins [2,3] that catalyze the nucleophilic displacement of L-methionine (L-met) from SAM with halides to form halogenated 5′-deoxyadenosine (5′-XDA). [4,5] These enzymes belong to a family of over 100 archaeal and bacterial proteins with no assigned function (pfam 01887, DUF62). Here we report that a DUF62 member from the recently sequenced marine bacterium Salinispora arenicola CNS-205 (SaDUF62, Sare_1364, genome accession number NC_ 009953) has no significant halogenase, but instead SAM hydrolase (adenosine-forming) activity in vitro.SAM is biosynthesized from ATP and L-met by SAM synthetase MetK. Besides its well-known and essential role as a methyl donor to nucleic acids and proteins, [6] the electrophilic character of the carbons surrounding the sulfonium group of SAM makes it also a source of methylene, amino, ribosyl, and aminoalkyl groups, as well as 5′-deoxyadenosyl radicals. [1] Furthermore, SAM acts as a molecular effector in the riboswitch-mediated feedback regulation of metK and methionine biosynthesis genes in bacteria [7,8] and plants. [9] Yet, the regulatory role of SAM is not limited to the met operon. SAM levels have been shown to influence morphological differentiation and secondary-metabolite biosynthesis in the soil bacteria Streptomyces-high levels of SAM cause antibiotic overproduction and inhibit sporulation-at least in part by promoting transcription of regulatory genes. [10][11][12] Correspondence to: Alessandra S. Eustáquio.
Note Added in ProofDuring revision of this manuscript, a similar study by Deng et al. [29] describing the in vitro characterization of PhDUF62 was published. The authors show that while PhDUF62 is unable to catalyze fluorination or chlorination reactions, it does convert SAM to adenosine in vitro. Assay of the enzyme in H 218 O results in [5′-18 O]adenosine without labeling of the protein; this indicates that a mechanism of the type depicted in Figure 2 is operating rather than a second possibility through an enzyme-bound intermediate.
HHMI Author Manuscript
HHMI Author Manuscript
HHMI Author ManuscriptWe recently reported that biosynthesis of the marine bacterial natural product salinosporamide A from Salinispora tropica involves a SAM-dependent chlorinase [5] similar to fluorinase from the fluoroacetate-producer Streptomyces cattleya. [4] These two enzymes represent yet another example of the versatility of SAM-binding proteins, [2,3] and are the only members characterized from a family of over 100 bacterial and archaeal homologues available in GenBank and assigned as "protein of unknown function, DUF62, pfam 01887". Given their wide distribution in the b...
The structures of the O-glycosyltransferase LanGT2 and the engineered,
C—C bond-forming variant LanGT2S8Ac show how the replacement of a single
loop can change the functionality of the enzyme. Crystal structures of the
enzymes in complex with a nonhydrolyzable nucleotide-sugar analogue revealed
that there is a conformational transition to create the binding sites for the
aglycon substrate. This induced-fit transition was explored by molecular docking
experiments with various aglycon substrates.
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