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On the catalytic activity of a GT1 family glycosyltransferase from Streptomyces venezuelae ISP5230 Stephanie M Forget, Sydney B Shepard, Ebrahim Soleimani, and David L. Jakeman J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.9b01130 • Publication Date (Web): 20 Aug 2019 Downloaded from pubs.acs.org on August 23, 2019
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The Journal of Organic Chemistry
On the catalytic activity of a GT1 family glycosyltransferase from Streptomyces venezuelae ISP5230 Stephanie M. Forget,a Sydney B. Shepard,b Ebrahim Soleimani,b,c and David L. Jakeman* a,b. a
Department of Chemistry, Dalhousie University; bCollege of Pharmacy, Dalhousie University; cDepartment of Chemistry, Razi University, Kermanshah 67149-67346, Iran.*
[email protected].
KEYWORDS. Glycosyltransferase, glycodiversification, chemo-enzymatic synthesis, CAZy enzyme, natural products, biocatalysis ABSTRACT: GT1-family glycosyltansferase, Sv0189, from Streptomyces venezuelae ISP5230 (ATCC 10721) was characterized. The recombinantly produced protein Sv0189 possessed UDP-glycosyltransferase activity. Screening, using an assay employing unnatural nitrophenyl glycosides as activated donors, resulted in the discovery of a broad substrate scope with respect to both acceptor molecules and donor sugars. In addition to polyphenols, including anthraquinones, simple aromatics containing primary or secondary alcohols, a variety of complex natural products and synthetic drugs were glucosylated or xylosylated by Sv0189. Regioselectivity was established through the isolation and characterization of glucosylated products. Sv0189 and homologous proteins are widely distributed amongst Streptomyces species and their apparent substrate promiscuity reveals potential for their development as biocatalysts for glycodiversification.
Introduction Glycosylation of bacterial secondary metabolites is a common structural modification with data suggesting that 20% are glycosylated.1 The addition of a sugar alters the physicochemical and biological properties of the aglycone, potentially modulating solubility, targetrecognition, specificity, pharmacology and toxicity.1-4 In recent years, a growing number of glycosyltransferScheme 1. General reactivity of GT1 family glycosylases have been shown to be promiscuous towards transferases using UDP-Glc as a donor. sugar donors.5-11 Harnessing these reactions holds great potential for the development of regiospecific, green catalysts for application in late-stage functionalStrikingly, several glycosyltransferases have been ization of complex molecules. Enzymes offer key adshown to possess relaxed specificity towards acceptor vantages over synthetic methods, including their biocompounds. The most extensively studied of these is degradability and high catalytic efficiency at ambient OleD, which has been evolved, in multiple iterations, temperatures under aqueous conditions. The glycosylto further expand its substrate scope towards both dotransferases implicated in small molecule secondary nors and acceptors.15, 16 The GT1 family glycosyltransmetabolism are predominantly members of the GT1 12, 13 ferase Sv0189 found within the genome of Streptomyfamily (CAZy database); these Leloir-type glycosylces venezuelae was identified as a close homologue of transferases utilize sugar nucleotides as donors from OleD (75 % identity).17-19 Homologous proteins to which the sugar is transferred to an acceptor molecule Sv0189 are highly conserved across Streptomyces spewith concomitant release of a nucleoside diphosphate. cies, and many are not associated with discrete secSugar transfer is achieved by an inverting mechanism, ondary metabolite biosynthetic gene clusters (BGCs). facilitated by a catalytic base, to yield a b-glycosylated Herein, we recombinantly express and purify Sv0189, product from an a-configured sugar nucleotide as characterize its activity as a glycosyltransferase, and shown in Scheme 1.14 ACS Paragon Plus Environment
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perform a substrate scope screen with a diverse library comprising 96 potential acceptors, including phenols, anthraquinones, natural products, and synthetic drugs. Sv0189 was also probed for its ability to utilize alternate sugar donors. Several glycosylation products were isolated to delineate regioselective glycosylation. Results and discussion Sv0189 Genomic Context Evaluation of the genomic context of sv0189 (GenBank: CCA53477.1) did not provide insight into the physiological function of glycosyltransferase Sv0189. In a previous study, the Streptomyces mutant bearing deletion of sv0189 was found to grow robustly, with an unaltered phenotype in reference to the wild-type strain.20 Analysis of the S. venezuelae ATCC10721 (ISP5230) genome,21 using antiSMASH, a bioinformatics tool used to predict putative BGCs,22 did not situate sv0189 within a BGC. While OleD is associated with inactivation of macrolide antibiotics, no predicted macrolide BGCs were identified within the S. venezuelae ATCC 10721 genome. AntiSMASH detected 84 predicted secondary metabolite BGCs (Table S1) in the genome, eight of which have been linked to characterized natural products including chloramphenicol (cluster 14), the jadomycins (cluster 67), gaburedin (cluster 46), venezuelin (cluster 10), foroxymithine (cluster 80), (+)-isodauc-8-en-11-ol (cluster 9), venemycin (cluster 8a), and isopyochelin (cluster 8b).23-29 sv0189 was not found within a predicted BGC, although it was situated in close proximity to cluster 3, which possesses low (6 %) gene similarity to a number of disparate secondary BGCs including those encoding hygromycin B (saccharide), herbimycin (macrolide), collismycin A (2,29-bipyridyl), yatakemycin (cyclopropapyrroloindole) and alnumycin (napthoquinone). Therefore there is no clear evidence that Sv0189 is associated with a secondary metabolite gene cluster. Sv0189 Homologous proteins A protein Blast using Sv0189 as the query returned 500 proteins with sequence identity of greater than 55 %. Given the widespread distribution of highly conserved proteins, we posit that a general function in Streptomyces is more plausible than a role in secondary metabolism, also consistent within the genomic context. The protein sequence of Sv0189 showed high sequence identity (75 %) to OleD (Figure S1), an enzyme known to possess relaxed substrate tolerance and, therefore, Sv0189 was also predicted to possess a similarly relaxed tolerance warranting further investigation.15 OleD has been linked to host-resistance for its ability to transfer glucose to oleandomycin and a host of other macrolide antibiotics including, carbocin, tylosin, and erythromycin, conferring protection to the producing strain S. antibioticus.17, 30 However, given the presence of OleI, a glycosyltransferase with enhanced specificity towards oleandomycin that is directly encoded within
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the oleadomycin BGC, OleD has been proposed to be involved more generally in xenobiotic resistance.30 Sv0189 also possessed high sequence identity to the characterized macrolide inactivation glycosyltransferases, MGT, from Streptomyces lividans TK21 (76%) and Streptomyces ambofaciens ATCC 23877 (74%).31-33 An unrooted phylogenetic comparison of these proteins (OleD, OleI, MGT from each S. lividans and S. ambofaciens, and Sv0189) showed OleI to be most distantly related, while OleD and the other characterized MGTs were more closely related to each other than to Sv0189 (Figure S2). Analysis of the sequence alignments were carried out to probe whether amino acid differences between Sv0189 occurred at positions important for substrate recognition. Key positions were drawn from previous studies on OleD, allowing for comparison of residues known to be implicated in substrate binding.15, 34 Several features emerged that supported the hypothesis of a divergent substrate scope for Sv0189. Evolved variants of OleD consistently possess the mutation S132F, a residue involved in sugar binding, while in Sv0189 the equivalent position is already an aromatic residue, tryptophan (W134). A second difference of note is the presence of an aspartic acid at D70, which is located in a variable loop region associated with substrate binding, whereas for wild-type OleD this position is a proline (P67) and a threonine in evolved variants.35 The comparable residue for MGT from both S. lividans and S. ambofacien possess serine or proline, respectively. Establishment of glycosyltransferase activity and kinetics Recombinant Sv0189 bearing an N-terminal His6-tag was soluble after purification. Glycosyltransferase activity was first asessed through reaction of donor, 2chloro-4-nitrophenyl-β-D-glucopyranoside (2ClPNPGlc) and uridine 5ʹ-diphosphate (UDP). The use of unnatural nitrophenyl glycosyl donors has been well established for the study of glycosyltransferases.36 Transfer of glucose to UDP by Sv0189, releasing 2chloro-4-nitro-phenolate (2ClPNP), was monitored by UV detection and observed qualitatively through evolution of a yellow colour. The product uridine 5ʹ-diphosphate a-D-glucose (UDP-Glc) was observed by high resolution mass spectrometry (HRMS, Table S2). In the absence of Sv0189, yellow colour evolution was not observed over the monitored time-course. Enzyme rates were measured in different buffers and a different pH values in order to optimize the reaction (Figure S3), resulting in the selection of phosphate buffered saline (PBS) buffer (50 mM) at pH 7.0 for all subsequent reactions. The protein remained soluble in tris(hydroxymethyl)aminomethane (TRIS) or PBS buffers (50 mM, pH 7.0) at 4°C for at least a week, with no observed loss of activity; however, only 10% of enzyme activity remained after a freeze-thaw cycle.
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The Journal of Organic Chemistry
The Michaelis-Menten kinetic parameters for Sv0189 were determined using UDP or deoxythymidine 5ʹ-diphosphate (dTDP), and 2ClPNPGlc and are presented in Table 1 (see also Figures S6 – S8). The Km value for UDP, 0.09 mM, was comparable to that found for OleD, 0.25 mM, and was consistent with UDP-Glc as a physiological substrate. While the kcat and kcat/Km values were relatively low, this was attributed primarily to the low affinity (Km 1.5 mM) of the donor, 2ClPNPGlc.37 This high Km value of the donor is not surprising given that 2ClPNPGlc is not the natural substrate, which is unknown. The tolerance of Sv0189 towards other pyrimidine and purine nucleosides was also evaluated. UDP was found to be the preferred substrate, with 1900-fold better efficiency compared to dTDP. The Km for dTDP is greater than for UDP and the kcat is decreased, both by approximately one order of magnitude and therefore decreasing the overall catalytic efficiency. This contrasts the kinetics observed for wild-type OleD, which prefers UDP over dTDP by a factor of approximately ten in terms of catalytic efficiency, with the difference being driven by the kcat and not by Km.37 No activity was detected with the purine nucleosides tested, adenine 5ʹ-diphosphate (ADP) or guanidine 5ʹ-diphosphate (GDP). Table 1. Sv0189 kinetic parameters Variable substrate UDPa ClPNPGlcb dTDPa Km (mM) 0.091 1.5 ±0.06 1.7 ±0.2 ±0.008 kcat (min-1) 7.0 ±0.2 8.9 ±0.1 0.07 ±0.003 kcat/Km(mM76.8 ±0.1 5.9 ±0.04 0.04 ±0.1 1 min-1) a ClPNPGlc (5 mM) or bUDP (2.5 mM) as the saturating substrate. Acceptor Scope Screening A library of 96 acceptors (Figure S9) was screened using an assay employing 2ClPNPGlc.15 A diverse set of putative acceptors was selected to probe the reaction scope, including simple alcohols and polyphenol (118), natural products (19-70,95-96) and synthetic drugs (71-94). The selected molecules were selected to survey a wide variety of potential templates, including primary and secondary alcohols, amines, thiols, and more complex natural product scaffolds including macrolides, anthracyclines, polyenes, peptides, alkaloids and oligosaccharides. Several synthetic drugs were selected, which feature a variety of potential glycosylation sites including stereo-defined secondary alcohols, tertiary alcohols, secondary thiol and secondary and tertiary amines. The assay employed a catalytic concentration of UDP, which, on reaction with 2ClPNPGlc, released phenolate 2ClPNP with concomitant production of UDP-Glc
in situ (Scheme 2). Since production of phenolate requires consumption of limiting UDP-Glc, phenolate production serves as a proxy for acceptor glycosylation. The assay was performed in a 96-well plate, such that initial reaction rates could be monitored by UV absorbance. After a 48 h incubation period, reactions were quenched and new products identified by HPLC and by LC-HRMS. The rate observed with acceptor 1 (4-methylumbelliferone), having been established as an acceptor in preliminary screening, of 5 mAu/min served as a benchmark for a positive hit, while a reaction containing 10 % DMSO with no acceptor provide a benchmark for a negative result (
