Enzymatic Synthesis of the Ribosylated Glycyl-Uridine Disaccharide Core of Peptidyl Nucleoside Antibiotics

Article abstract of DOI:10.1021/acs.joc.8b00855

Muraymycins belong to a family of nucleoside antibiotics that have a distinctive disaccharide core consisting of 5-amino-5-deoxyribofuranose (ADR) attached to 6′-N-alkyl-5′-C-glycyluridine (GlyU). Here, we functionally assign and characterize six enzymes from the muraymycin biosynthetic pathway involved in the core assembly that starts from uridine monophosphate (UMP). The biosynthesis is initiated by Mur16, a nonheme Fe(II)- and α-ketoglutarate-dependent dioxygenase, followed by four transferase enzymes: Mur17, a pyridoxal-5′-phosphate (PLP)-dependent transaldolase; Mur20, an aminotransferase; Mur26, a pyrimidine phosphorylase; and Mur18, a nucleotidylyltransferase. The pathway culminates in glycosidic bond formation in a reaction catalyzed by an additional transferase enzyme, Mur19, a ribosyltransferase. Analysis of the biochemical properties revealed several noteworthy discoveries including that (i) Mur16 and downstream enzymes can also process 2′-deoxy-UMP to generate a 2-deoxy-ADR, which is consistent with the structure of some muraymycin congeners; (ii) Mur20 prefers l-Tyr as the amino donor source; (iii) Mur18 activity absolutely depends on the amine functionality of the ADR precursor consistent with the nucleotidyltransfer reaction occurring after the Mur20-catalyzed aminotransfer reaction; and (iv) the bona fide sugar acceptor for Mur19 is (5′S,6′S)-GlyU, suggesting that ribosyltransfer occurs prior to N-alkylation of GlyU. Finally, a one-pot, six-enzyme reaction was utilized to generate the ADR-GlyU disaccharide core starting from UMP.

Full text of DOI:10.1021/acs.joc.8b00855

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Article
Enzymatic Synthesis of the Ribosylated Glycyl-Uridine
Disaccharide Core of Peptidyl Nucleoside Antibiotics
Zheng Cui, Xiaodong Liu, Jonathan Overbay, Wenlong Cai, Xiachang Wang, Anke Lemke,
Daniel Wiegmann, Giuliana Niro, Jon S Thorson, Christian Ducho, and Steven G. Van Lanen
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b00855 • Publication Date (Web): 17 May 2018
Downloaded from http://pubs.acs.org on May 17, 2018
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Enzymatic Synthesis of the Ribosylated GlycylꢀUridine Disaccharide Core of Peptidyl
Nucleoside Antibiotics
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Zheng Cui,† Xiaodong Liu,† Jonathan Overbay,† Wenlong Cai,† Xiachang Wang,‡,§ Anke
Lemke,⊥ Daniel Wiegmann,⊥ Giuliana Niro,⊥ Jon S. Thorson,†,‡ Christian Ducho,⊥ and Steven G.
Van Lanen*†
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†Department of Pharmaceutical Sciences and Center for Pharmaceutical Research and
‡
Innovation, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536, United
States
§Jiangsu Key Laboratory for Functional Substance of Chinese Medicine, School of Pharmacy,
Nanjing University of Chinese Medicine, Nanjing 210023, People’s Republic of China
⊥Department of Pharmacy, Pharmaceutical and Medicinal Chemistry, Saarland University,
Campus C2 3, 66123 Saarbrücken, Germany
*To whom correspondence should be addressed. Tel: 1ꢀ859ꢀ323ꢀ6271; Email:
svanlanen@uky.edu
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ABSTRACT
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Muraymycins belong to a family of nucleoside antibiotics that have a distinctive disaccharide
core consisting of 5ꢀaminoꢀ5ꢀdeoxyribofuranose (ADR) attached to 6′ꢀNꢀalkylꢀ5′ꢀCꢀ
glycyluridine (GlyU). Here we functionally assign and characterize six enzymes from the
muraymycin biosynthetic pathway involved in the core assembly that starts from UMP. The
biosynthesis is initiated by Mur16, a nonꢀheme Fe(II)ꢀ and αꢀketoglutarateꢀdependent
dioxygenase, followed by four transferase enzymes—Mur17, a pyridoxalꢀ5′ꢀphosphate (PLP)ꢀ
dependent transaldolase; Mur20, an aminotransferase; Mur26, a pyrimidine phosphorylase; and
Mur18, a nucleotidylyltransferase. The pathway culminates in glycosidic bond formation in a
reaction catalyzed by an additional transferase enzyme, Mur19, a ribosyltransferase. Analysis of
the biochemical properties revealed several noteworthy discoveries including that (i) Mur16 and
downstream enzymes can also process 2′ꢀdeoxyꢀUMP to generate a 2ꢀdeoxyꢀADR, which is
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consistent with the structure of some muraymycin congeners; (ii) Mur20 prefers LꢀTyr as the
amino donor source; (iii) Mur18 activity absolutely depends on the amine functionality of the
ADR precursor, consistent with the nucleotidyltransfer reaction occurring after the Mur20ꢀ
catalyzed aminotransfer reaction; and (iv) the bona fide sugar acceptor for Mur19 is (5′S,6′S)ꢀ
GlyU, suggesting that ribosyltransfer occurs prior to Nꢀalkylation of GlyU. Finally, a oneꢀpot,
sixꢀenzyme reaction was utilized to generate the ADRꢀGlyU disaccharide core starting from
UMP.
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INTRODUCTION
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Several structural classes of nucleoside antibiotics have been discovered as a consequence of
their ability to inhibit translocase I (MraY), an essential bacterial enzyme that initiates the lipid
cycle of peptidoglycan.1ꢀ3 One class is characterized by a disaccharide core consisting of a 5ꢀ
aminoꢀ5ꢀdeoxyribofuranose (ADR) attached to 6′ꢀNꢀalkylꢀ5′ꢀCꢀglycyluridine (GlyU) through a
standard βꢀOꢀglycosidic bond, exemplified by the structurally simplest member, FRꢀ900493 (1)
from Bacillus cereus (Figure 1).4 Other members of this structural class, which notably differ in
the alkyl substituent of the GlyU, are represented by the caprazamycins (represented by
liposidomycin LꢀI (2) from Streptomyces sp. SNꢀ1061M,5,6 caprazamycin B (3) from
Streptomyces sp. MK730ꢀ62F2,7,8 Aꢀ90289 B (4) from Streptomyces sp. SANK 60405,9 and
muraminomicin F (5) from Streptosporangium amethystogenes10), sphaerimicins [represented by
sphaerimicin A (6) from Sphaerisporangium sp. SANK 60911],11 and muraymycins [represented
by muraymycin C1 (7) from Streptomyces sp. NRRL 30471].12 The producing strains of 2ꢀ7
biosynthesize several congeners that vary in the length and functionality of the fatty acyl side
chain or by the substitution pattern of the ADRꢀGlyU disaccharide core. As an example of the
latter, the isolated muraymycins
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Figure 1. Structure of representative nucleoside antibiotics containing an ADRꢀGlyU disaccharide core.
can contain a methylated ADR as in 7, a standard 2′′ꢀOH variant [exemplified with muraymycin
C2 (8)], or a 2′′ꢀdeoxy variant, exemplified by muraymycin C3 (9).12ꢀ14
The biosynthetic mechanism for GlyU and ADR has previously been defined using the
enzymes involved in the biosynthesis of 4 (Figure 2).15ꢀ18 Both components start from UMP
(10a) in a reaction catalyzed by LipL, a nonꢀheme Fe(II), αꢀketoglutarate (αKG)ꢀdependent
dioxygenase that catalyzes an unusual oxidative dephosphorylation via stereospecific 5′ꢀ
hydroxylation of 10a to yield uridineꢀ5′ꢀaldehyde (11a).16,19 The two pathways diverge after the
formation of 11a. In one pathway, a pyridoxalꢀ5′ꢀphosphateꢀdependent (PLP)
LꢀThr:11a
transaldolase LipK catalyzes the stereospecific generation of the nonproteinogenic βꢀhydroxy
amino acid, 5′S,6′SꢀGlyU (12).17 In the other pathway, a distinct PLPꢀdependent enzyme LipO
initiates the synthesis of ADR by catalyzing the transamination of 11a to 5′ꢀaminoꢀ5′ꢀ
deoxyuridine (13a) using
L
ꢀmethionine as the amino group donor.18 The nucleoside
phosphorylase LipP subsequently generates uracil (14) and 5ꢀaminoꢀ5ꢀdeoxyꢀαꢀ
D
ꢀriboseꢀ1ꢀ
phosphate, which is activated to UDPꢀ5ꢀaminoꢀ2,5ꢀdideoxyribose (15a) in a reaction catalyzed
by the nucleotidyltransferase, LipM. Although this mechanism of sugar activation, i.e. generation
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of an NDPꢀsugar, is standard during the biosynthesis of glycosylated natural products, a unique
feature of LipM is the strict selectivity toward the amine functionality of the ribofuranose
substrate. Finally, using uridine as a surrogate sugar acceptor, the ribosyltransferase LipN was
shown to catalyze the formation of the βꢀOꢀglycosidic bond of the unnatural disaccharide 16. By
extension, LipN was proposed to catalyze the formation of the ADRꢀGlyU disaccharide 17a
using the more hindered LipKꢀproduct 12 as an acceptor, although this remains to be verified.
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As expected, the biosynthetic gene clusters for 2, 3, and 5ꢀ7 encode for homologous
Figure 2. Biosynthesis of the ADRꢀGlyU disaccharide core 17a. Enzymes from the 4 biosynthetic pathway
(annotated as Lip proteins in bold) were previously functionally assigned using the probable native substrates with
the lone exception being LipN, which was characterized using a surrogate sugar acceptor, uridine. The Mur proteins
involved in 7ꢀ9 biosynthesis have moderate sequence homology to the corresponding Lip protein and hence
putatively catalyze the same reaction.
proteins involved in the biosynthesis of the ADRꢀGlyU disaccharide of 4.10,11,20ꢀ22 For
muraymycins this includes mur16 (encoding a putative αKG:10a dioxygenase related to LipL),
mur17 (LꢀThr:11a transaldolase, LipK), mur20 (LꢀMet:11a aminotransferase, LipO), mur26 (13a
phosphorylase, LipP), mur18 (nucleotidylyltransferase, LipM) and mur19 (15a:12
ribosyltransferase, LipN), with sequence identities ranging from 34% to 46% based on pairwise
alignments (Figure S1).22 Unlike in 4, the muraymycin disaccharide core is not modified with a
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2′ꢀOꢀsulfate and, as previously noted, the 2′′ꢀOH of the ADR is differentially modified,
suggesting the possibility that the homologous proteins have unique biochemical properties that
direct these structural variations. In this paper, the function and substrate specificity for Mur16ꢀ
20 and Mur26 are defined using recombinant enzymes in vitro, and the results compared with the
homologs involved in 4 biosynthesis. Additionally, the ribosyltransferase Mur19 from the 7ꢀ9
biosynthetic pathway and LipN from the 4 biosynthetic pathway were tested, for the first time,
with the hypothesized genuine ribosyl acceptor, 12. Finally, based on information garnered from
substrate specificity studies, a oneꢀpot enzymatic reaction was employed to produce 17a and its
2″ꢀdeoxy analogue.
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RESULTS AND DISCUSSION
Mur16, a nonꢀheme, Fe(II)ꢀdependent αKG:10 dioxygenase. The mur16 gene was cloned
and expressed in Escherichia coli BL21(DE3) to yield soluble protein (Figure 3A). The activity
of Mur16 was tested with 10a under optimized conditions previously reported for LipL.16 Using
HPLC for analysis, a new peak appeared that coꢀeluted with the product of the LipLꢀcatalyzed
reaction and synthetic uridineꢀ5′ꢀaldehyde, 11a (Figure 3B).16 The identity of the product was
further supported by LCMS analysis, yielding an (M + H)+ ion at m/z = 243.1 and an (M + H3O)+
ion at m/z = 261.1, which are consistent with the molecular formula for 11a [expected (M + H)+
and (M + H3O)+ ions at m/z = 243.1 and 261.1, respectively, for C9H10N2O6] (Figure S2A).
Similar to LipL, conversion of 10a to 11a was only detected when αKG and FeCl2 were
included, and the activity was stimulated by the inclusion of ascorbate (Figure S2B). Mur16 was
subsequently determined to have optimal activity with 40 µM FeCl2 and 2 mM ascorbate. Based
on LCMS analysis, succinate was identified as a coꢀproduct (Figure S2C), which was indirectly
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supported by monitoring the oxidation of NADH using an enzymeꢀcoupled assay (Figure S2D).
Similar to several other enzymes of this dioxygenase superfamily, succinate was formed in the
absence of 10a via uncoupled oxidative decarboxylation of αKG, although the rate of formation
was significantly enhanced when 10a was included (Figure S2D). Mur16 was previously
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proposed to initiate the biosynthesis of
However, incubation of Mur16 with ꢀArg did not yield a product based on LCMS analysis, nor
was the rate of succinate formation increased when compared to the uncoupled oxidation
Lꢀepiꢀcapreomycidine of 7ꢀ9 by oxidation of L
ꢀArg.22
L
reaction control (Figure S2D). Thus, in contrast to prior predictions,
LꢀArg is not a substrate for
Mur16 under the conditions tested.
O
NH
O
O
H
H
O
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P
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HO
HO
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R
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Figure 3. Characterization of Mur16. (A) Reaction catalyzed by Mur16. (B) HPLC analysis of 12ꢀh incubations of
10a with (i) no enzyme and (ii) Mur16. (C) HPLC analysis of 12ꢀh incubations of 10b with (i) no enzyme and (ii)
Mur16. (D) HPLC analysis of 12ꢀh incubations of 10c with (i) no enzyme and (ii) Mur16. (E) Singleꢀsubstrate
kinetic analysis for the indicated substrate with near saturating αKG and 40 nM Mur16. A260, absorbance at 260 nm;
αKG, αꢀketoglutarate; succ, succinate.
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The substrate specificity of Mur16 was next examined. Using HPLC for detection, 2′ꢀdeoxyꢀ
UMP (10b) was revealed to be a substrate for Mur16 (Figure 3C), generating the respective
aldehyde product 11b based on LCMS analysis (Figure S2E). The hypothetical substrate 2′ꢀ
methoxyꢀUMP (10c) was synthesized,23 and the identity was confirmed by HRMS and NMR
spectroscopy (Figures S3 and S4). HPLC analysis of the Mur16ꢀcatalyzed reaction with 10c
revealed a new peak (Figure 3D), and LCMS analysis was consistent with the aldehyde product
11c (Figure S2F). The results for Mur16 with 10b and 10c are in contrast to LipL, which is
highly specific for 10a,16 but are entirely consistent with the isolation of the different
muraymycin congeners represented by 7ꢀ9. Singleꢀsubstrate kinetic analysis of Mur16 with 10a
and 10b revealed typical MichaelisꢀMenten kinetics, yielding kinetic constants of Km = 21 ± 4
µM and kcat = 4.4 ± 0.3 sꢀ1 with respect to 10a and Km = 6.3 ± 2.2 µM and kcat = 2.3 ± 0.2 sꢀ1 with
respect to 10b (Figure 3E). Comparison of the second order rate constants (2.1 × 105 Mꢀ1sꢀ1 and
3.7 × 105 Mꢀ1sꢀ1, respectively) suggests a lack of preference for either 10a or 10b. Singleꢀ
substrate kinetic analysis with 10c revealed nonꢀMichaelis Menten kinetics with apparent
inhibition at increasing substrate concentration (Figure 3E); however, the data did not fit well to
a kinetic equation describing simple substrate inhibition. Consequently, the specific activities at
the observed maximal velocity were compared, which revealed ≥ 15ꢀfold lower activity with 10c
compared to the other substrates. Thus, the kinetic results suggest that methylation of ADR
occurs following the Mur16ꢀcatalyzed reaction.
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Mur17, a PLPꢀdependent
LꢀThr:11a transaldolase. The mur17 gene was cloned and
expressed in Streptomyces lividans TK24 to yield soluble protein (Figure 4A). UVꢀVIS
spectroscopic analysis of recombinant Mur17 revealed a detectable UV maximum at 415 nm
(Figure S5A), suggesting that a minor fraction of the protein copurified with PLP as the internal
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aldimine. Using HPLC for detection, activity tests with exogenously supplied PLP and potential
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substrates LꢀThr and 11a revealed a new peak (Figure 4B). The formation of the new peak was
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significantly reduced without the addition of PLP (Figure S5B), which is consistent with the
UVꢀVIS spectroscopic analysis and the necessity of PLP in catalysis as previously reported for
LipK.17 LCMS analysis of the purified, new peak yielded an (M + H)+ ion at m/z = 318.1 (Figure
4B), which is consistent with the molecular formula for GlyU (12) [expected (M + H)+ ion at m/z
= 318.1 for C11H15N3O8]. As expected, the new peak coꢀeluted with the product of LipK as well
as with authentic 5′S,6′Sꢀ12,24ꢀ26 which is the relative stereochemistry that is observed in the
ADRꢀGlyU disaccharide core of muraymycins.12 Mur17 did not utilize 11b or 11c as substrates,
results of which are consistent with the structure of all known muraymycin congeners that have
the identical, ‘hydroxylated’ 12 nucleoside. To provide evidence for the stereochemical
assignment of 12, authentic 5′S,6′Sꢀ12 as well as its two 5′ꢀ and 6′ꢀepimers (5′R,6′Sꢀ12 and
5′S,6′Rꢀ12) were synthesized,24,25,27 and each isomer was modified with 6ꢀaminoquinolylꢀNꢀ
hydroxysuccinimidyl
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H2N
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Figure 4. Characterization of Mur17. (A) Reaction catalyzed by Mur17. (B) HPLC analysis of 12ꢀh incubation of
11a with (i) no enzyme and (ii) Mur17. The inset depicts the mass spectrum for the ion peak eluting at t = 5.6 min
corresponding to 12. (C) HPLC analysis of 6ꢀaminoquinolylꢀNꢀhydroxysuccinimidyl carbamate (AQC)ꢀmodified
(5′S,6′S)ꢀ12 after 12ꢀh incubation of 11a with (i) no enzyme and (ii) Mur17. The inset depicts the mass spectrum for
the ion peak eluting at t = 16.2 min corresponding to AQCꢀmodified (5′S,6′S)ꢀ12 [expected (M + H)+ ion at m/z =
488.1 for C21H21N5O9]. Under these HPLC conditions, AQCꢀmodified (5′S,6′R)ꢀ12 and (5′R,6′S)ꢀ12 elute at t = 19.5
min and t = 25.1 min, respectively, and have similar mass spectra as shown for AQCꢀmodified (5′S,6′S)ꢀ12. A260
,
absorbance at 260 nm.
carbamate (AQC) to generate the 6ꢀquinolinylaminocarbonyl amines without alteration of the
stereochemistry. LCMS comparison of the AQCꢀmodified isomers in comparison to the AQCꢀ
modified Mur17 product was consistent with stereospecific formation of 5′S,6′Sꢀ12 (Figure 4C).
Functional assignment of Mur20 as an LꢀTyr:11ꢀaminotransferase. The mur20 gene was
cloned and expressed in E. coli BL21(DE3) to yield soluble protein (Figure 5A). The successful
production of Mur20 in E. coli is in contrast to prior results with the homologous protein LipO,
which was only soluble upon production in Streptomyces lividans TK64.18 Using HPLC for
detection, activity tests with potential substrates 11a and LꢀMet revealed a new peak with an
identical retention time to the LipO product as well as synthetic 5′ꢀaminoꢀ5′ꢀdeoxyuridine (13a)
standard (Figure 5B).18 The identity of the product was further supported by LCMS analysis
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(Figure S6A), yielding an (M + H)+ ion at m/z = 244.0902, which is consistent with the
molecular formula for 13a [expected (M + H)+ ion at m/z = 244.0855 for C9H13N3O5]. The
expected coꢀproduct—αꢀketoꢀγꢀmethylthiol butyrate—was also detected by LCMS following
derivatization of the Mur20ꢀreaction components with 3ꢀmethylꢀ2ꢀbenzothiazolinone hydrazone
hydrochloride (MBTH) to give a mixture of cis and trans imine adducts (Figure 5C).28
10
11
12
13
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15
16
17
18
19
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O
O
H
O
O
N
NH
N
NH
H2N
O
O
O
NH2
COOH
O
HO
R
HO
R
X
X
COOH
CH3
N
S
R
R
N
N
C
R = CH2CH2SCH3 or CO2
H
Figure 5. Characterization of Mur20. (A) Reaction catalyzed by Mur20. (B) HPLC analysis of 6ꢀh incubation of
Met and 11a with (i) no enzyme, (ii) Mur20, and (iii) LipO. (C) HPLC analysis of MBTHꢀmodified components of
6ꢀh incubation of ꢀMet and 11a with (i) no enzyme, (ii) Mur20, and (iii) LipO. A260, absorbance at 260 nm.
Lꢀ
L
In contrast to Mur17 (Figure S5A) and LipO, the latter of which coꢀpurified with near
stoichiometric amounts of PLP based on UVꢀVIS spectroscopic analysis,18 recombinant Mur20
did not have clear a diagnostic absorption feature above 300 nm that would suggest
copurification with PLP (Figure S7A). Instead, a shoulder was evident at 363 nm within a long
tail that proceeded to around 500 nm. Given the established PLPꢀdependency of LipO and other
transaminases along with this inconclusive UVꢀVIS profile, we initially added PLP to the
Mur20ꢀcatalyzed reactions. Upon further examination, however, 13a was shown to be generated
in reactions without exogenously supplied PLP (Figure S7B). The activity under these
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conditions was 1.5ꢀ to 3ꢀfold decreased depending upon the batch of purified Mur20. Sequential
addition of substrates to Mur20 did not alter the UVꢀVIS spectrum (Figure S7A). As a
consequence of these results, which are similar to that previously reported for glutamateꢀglycine
transaminase,29 we can only conclude that PLP stimulates transamination at this time. Further
experimental analysis will be needed to ascertain a definitive role for PLP in Mur20ꢀmediated
catalysis.
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11
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19
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The substrate specificity of Mur20 toward different amine acceptors and donors was next
examined. Activity tests with 11b or 11c, the latter generated in situ by a Mur16ꢀcatalyzed
reaction starting from 10c, revealed both were amine acceptors (Figure S6B, C). Therefore, the
timing of Oꢀmethylation during muraymycin biosynthesis could not be discerned based on the
characterization of Mur20 along with consideration of the aforementioned Mur16 results. With
respect to amine donors, LꢀMet was initially utilized with Mur20 since LipO exhibited the
highest specific activity with this donor.18 Nonetheless, other potential amine donors—including
the remaining proteinogenic amino acids—were tested with Mur20. HPLC analysis revealed
several amine donors were readily utilized by Mur20 with 11a as the amine acceptor (Table S1).
A comparable broad selectivity toward the amine donor was previously noted for LipO. In
contrast to LipO, however, the specific activity for Mur20 was highest using
donor, followed closely by ꢀTrp, ꢀArg, ꢀMet, and AdoMet (Table S1). Comparison of the
specific activities revealed a ~2.6ꢀfold higher activity for LipO with ꢀMet compared to Mur20
with ꢀTyr.
LꢀTyr as an amine
L
L
L
L
L
Sugar activation by Mur26 and Mur18. The mur26 and mur18 genes were cloned and
expressed in E. coli BL21(DE3) to yield soluble proteins (Figure 6 and 7). The putative
phosphorylase activity of Mur26 was initially tested (Figure 6A). Using conditions that were
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employed during the functional assignment of the orthologous protein LipP,18 HPLC analysis
revealed that Mur26 catalyzed phosphotransfer using orthophosphate with coꢀsubstrate uridine,
13a, or 13b to generate uracil (14) and presumably the sugarꢀ1ꢀphosphates: αꢀDꢀriboseꢀ1ꢀ
phosphate, 5ꢀaminoꢀ5ꢀdeoxyꢀ αꢀDꢀriboseꢀ1ꢀphosphate, and 5ꢀaminoꢀ2,5ꢀdideoxyꢀαꢀDꢀriboseꢀ1ꢀ
phosphate, respectively (Figure 6B and C). Contrastingly, Mur26 did not utilize 13c, providing
the first, direct evidence to support that Oꢀmethylation occurs after formation of the sugarꢀ1ꢀ
phosphate.
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O
O
O
P
O
O
N
H2N
NH
H2N
OH
OH
NH
NH
O
O
HO
R
O
R
HO
Figure 6. Characterization of Mur26. (A) Reaction catalyzed by Mur26. (B) HPLC analysis of 6ꢀh incubations of
13a with (i) no enzyme, (ii) Mur26, and (iii) LipP. (C) HPLC analysis of 6ꢀh incubations of 13b with (i) no enzyme,
(ii) Mur26, and (iii) LipP. A260, absorbance at 260 nm.
The activity of the putative nucleotidyltransferase Mur18 was next assessed using sugarꢀ
1ꢀphosphates generated in situ by Mur26. Our previous analysis of the orthogous proteins LipP
and LipM revealed that the product of the tandem catalyzed reaction starting from 13a was
unstable, degrading to 10a and 5ꢀaminoꢀ5ꢀdeoxyꢀαꢀD
ꢀriboseꢀ1,2ꢀcyclophosphate (Figure 2).18
Contrastingly, the UDPꢀsugar was attainable when starting with the deoxy variant 13b, which
was utilized here with Mur26 and Mur18 (Figure 7A). Using HPLC for detection, analysis of
reactions containing both Mur26 and Mur18 revealed the formation of a new peak that was not
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detected in the controls (Figure 7B). A peak with the same retention time was observed when
either enzyme was substituted with the respective orthologous proteins LipP or LipM. LCMS
analysis of the new peak revealed an (M ꢀ H)ꢀ ion at m/z = 518.1 (Figure 7C), which is
9
10
11
12
13
14
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17
18
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consistent with the molecular formula for UDPꢀ5ꢀaminoꢀ2,5ꢀdideoxyꢀαꢀDꢀribose (15b) [expected
(M ꢀ H)ꢀ ion at m/z = 518.1 for C14H23N3O14P2]. Thus, the data are consistent with Mur26
producing 5ꢀaminoꢀ2,5ꢀdideoxyꢀαꢀDꢀriboseꢀ1ꢀphosphate, which serves as the substrate for
Mur18ꢀcatalyzed nucleotidyltransfer to yield 15b. Similar to the prior results reported for
LipM,17 no Mur18 activity was observed when starting from uridine, which is converted to αꢀ
riboseꢀ1ꢀphosphate by Mur26. Therefore, Mur18 and LipM constitute an unusual group of
nucleotidyltransferases that absolutely require an aminated sugarꢀ1ꢀphosphate for catalysis in
contrast to the standard hydroxylated ribose.
D
ꢀ
Functional assignment of Mur19 as a 15:12 ribosyltransferase. LipN was previously assigned
as a ribosyltransferase by utilizing uridine as a surrogate sugar acceptor and 15a—enzymatically
generated in situ starting from 13a—as a sugar donor.18 Here we aimed to identify the genuine
sugar acceptor for LipN and Mur19, in combination with either 15a or 15b as sugar donors. The
mur19 gene was cloned and expressed in S. lividans TK24 to yield soluble protein (Figure 8A).
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O
O
O
O
O
O
H2N
H2N
O
N
NH
O
P
OH
O
P
O
P
O
OH
OH
OH
HO
O
HO
HO
OH
8
9
10
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12
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100
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40
20
0
518.1
2
3
4
5
6
7
8
9
10
200
400
m/z
600
800
Elution Time (min)
Figure 7. Characterization of Mur18. (A) Reaction catalyzed by Mur26 (or LipP) and Mur18. (B) HPLC analysis of
6ꢀh incubations of 13b with (i) no enzymes, (ii) Mur26 or LipP, and (iii) LipP and Mur18. (C) Mass spectrum for
the ion peak eluting at t = 5.9 min corresponding to 15b. A260, absorbance at 260 nm.
In contrast to the results with LipN, Mur19 incubated with uridine and in situꢀgenerated 15a did
not yield a new product based on HPLC analysis. When uridine was substituted with authentic
5′S,6′Sꢀ12, which was established here as the product of Mur16 and Mur17 catalysis, HPLC
analysis of the reaction revealed a new peak (Figure 8B). The formation of the new peak was
dependent upon the inclusion of 5′S,6′Sꢀ12, 13a, Mur18, Mur26, and Mur19. LCMS analysis of
the new peak yielded an (M + H)+ ion at m/z = 449.1463 (Figure 8C), which is consistent with
the molecular formula for ADRꢀGlyU disaccharide (17a) [expected (M + H)+ ion at m/z =
449.1442 for C16H24N4O11]. To simplify the analytical identification of the product, 17a was
prepared by chemical synthesis (Figures S8ꢀS12);30 analysis of the Mur19ꢀcatalyzed reaction
revealed the new peak coꢀeluted with, and had identical UV and MS spectroscopic properties to,
authentic 17a. The data are therefore consistent with the functional assignment of Mur19 as a
12:15a 5ꢀaminoꢀ5ꢀdeoxyribosyltransferase.
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H2N
O
COOH
O
O
O
H
O
O
O
H2N
O
N
NH
N
NH
O
O
P
O
P
O
H2N
H
OH
OH
O
HO
OH
O
HO
OH
OH
HO
HO
OH
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Figure 8. Characterization of Mur19. (A) Reaction catalyzed by Mur19 with in situꢀgenerated 15a. (B) HPLC
analysis of 6ꢀh incubations of 12 and 13a with (i) Mur26; (ii) Mur26 and Mur18; and (iii) Mur26, Mur18, and
Mur19. Identical HPLC chromatograms were obtained upon substitution of Mur26 and Mur18 with the respective
ortholog involved in 4 biosynthesis. ADRꢀGlyU (17a) was prepared by chemical synthesis and used as a control. (C)
Mass spectrum for the ion peak eluting at t = 2.6 min corresponding to 17a. A260, absorbance at 260 nm.
The substrate selectivity of Mur19 was examined with respect to both sugar donor and
acceptor. For the former, Mur19 was incubated with 12 and in situꢀgenerated 15b (Figure
S13A), and HPLC analysis revealed a new peak that was not present in the controls (Figure
S13B). To identify the product, the reaction components were first modified with AQC, and the
peak corresponding to the AQCꢀderivatized product was purified. Both HRMS (Figure S13C)
and NMR spectroscopy (Figures S14 and S15) were consistent with the structure of AQCꢀ
modified 17b. The ability of Mur19 to transfer 2ꢀdeoxyribose to 12 is consistent with the
structures of some muraymycin congeners, exemplified by 9 (Figure 1). Likewise the inability
of LipN to transfer the 2ꢀdeoxyribose variant is consistent with the structure of known 4
congeners, all of which contain the standard 2ꢀhydroxylated aminoribosyl component.9
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The substrate selectivity for Mur19 with respect to the sugar acceptor was next
interrogated, primarily to decipher whether ribose attachment occurs prior to or after Nꢀ
alkylation of 12. Notably, muraymycins lacking the ADR component have been isolated from
the producing strain as minor metabolites,12,13 suggesting that ribosyltransfer could possibly
occur even later in the biosynthetic pathway, i.e., after peptide attachment. To entertain this
possibility, muraymycin D4, which lacks the ADR component, was isolated following standard
fermentation of the producing strain (Figure S16). However, incubation of Mur19 with in situ
generated 15a and muraymycin D4 did not yield a new product. We also explored the potential
reversibility of the Mur19ꢀcatalyzed reaction with the assumption that ribosylation occurs post
peptidation. Muraymycins D1, D2, and D3, which contain the ADR component with the same
modifications found in C1, C2, and C3, respectively, were isolated,14 and each congener was
incubated with excess UDP and Mur19. However, HPLC analysis of the reactions did not reveal
a new peak (Figure S17). Although negative results, the data with muraymycins D1ꢀD4 are
consistent with ribosylation happening before peptide attachment. To narrow down the timing of
the Mur19ꢀcatalyzed reaction, the hypothetical pathway intermediate aminopropylꢀ12 (18),
which lacks the ADR and peptide components, was synthesized and confirmed by HRMS and
NMR spectroscopic analyses (Figures S18ꢀS23). Similar to the results with muraymycin D4,
incubation of Mur19 with in situꢀgenerated 15a and 18 did not yield a new product. Therefore,
the totality of the data suggest that Mur19ꢀcatalyzed ribosylation occurs prior to Nꢀalkylation and
that 12 is the most likely substrate in vivo.
10
11
12
13
14
15
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18
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Glycosyltransferases are mechanistically classified as either inverting or retaining
enzymes based on the stereochemistry of the anomeric bond of the sugar before and after
formation of the glycoside product.31ꢀ33 Inverting glycosyltransferases are generally accepted to
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follow a single displacement mechanism, thereby requiring the formation of a ternary complex
during the reaction coordinate. Mur19 falls within the inverting glycosyltransferase
classification, suggesting that the donor 15 and acceptor 12 are bound simultaneously at the
active site preceding catalysis. Unlike typical inverting glycosyltransferases, which have
structurally distinct donors and acceptors,34 Mur19 utilizes two structurally similar, uridineꢀ
containing substrates, raising the question of how this ribosyltransferase differentially binds and
orients the two substrates for glycosidic bond formation. Mechanistic and structural
investigations for this enzyme are ongoing to interrogate the molecular details behind this
unusual substrate selection.
10
11
12
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Total Enzymatic Synthesis of 17a and 17b. We finally aimed to reconstitute the biosynthesis of
17a in vitro starting from the primary metabolite 10a using six enzymes: Mur16, Mur17, LipO,
LipP, Mur18 and Mur19. The enzymes were selected based upon their desirable properties such
as solubility when produced in E. coli (Mur18), substrate flexibility (Mur16 and Mur19), or
superior catalytic activity (LipO). With the addition of each enzyme, a new peak corresponding
to the expected product was detected by HPLC, including a small, new peak corresponding to
17a when all enzymes were present (Figure 9A). Yields of 17a were low under these conditions
(5% with respect to 10a) with a significant amount of the enzymatically generated sugar acceptor
12 remaining after termination of the reaction. Similarly, using 10b and 12 as starting material,
the production of 17b was attempted using five enzymes: Mur16, LipO, LipP, Mur18 and
Mur19. In this case, the exogenous supply of 12 was essential given that Mur17 cannot utilize
11b, the product of Mur16. HPLC analysis of the fiveꢀenzyme reaction revealed complete
consumption of 12 with the formation of the expected product, 17b (Figure 9B).
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The chemical synthesis of ADRꢀGlyU has previously been established starting from uridine
5
6
7
8
(acceptor) and
D
ꢀribose (donor).30,35,36 This procedure involves eight linear steps to the final
ꢀribose, with convergence of the acceptor and donor
product with respect to either uridine or
D
9
10
11
12
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halfway through the sequence. An overall yield of ~3.5% with respect to the starting reagent
uridine has been achieved in our hands following this synthetic scheme.30 Here the synthesis of
17a starting from 10a was accomplished in a single pot, sixꢀenzyme reaction with comparable
yields. Thus, without any optimization, the enzymatic synthesis appears to be on par with the
chemical route.
Figure 9. Biosynthesis of ADRꢀGlyU. (A) HPLC analysis of oneꢀpot reaction starting with 10a and UTP (*impurity
in commercial UTP) after 12ꢀh incubation with (i) no enzymes; (ii) Mur16; (iii) Mur16 and Mur17; (iv) Mur16,
Mur17, and LipO; (v) Mur16, Mur17, LipO, and LipP; (vi) Mur16, Mur17, LipO, LipP, and Mur18; (vii) Mur16,
Mur17, LipO, LipP, Mur18, and Mur19; and (viii) 17a standard. (B) HPLC analysis of oneꢀpot reaction starting with
10b, UTP, and 12 after 12ꢀh incubation with (i) no enzymes; (ii) Mur16; (iii) Mur16 and LipO; (iv) Mur16, LipO,
and LipP; (v) Mur16, LipO, LipP, and Mur18; (vi) Mur16, LipO, LipP, Mur18, and Mur19. A260, absorbance at 260
nm.
A considerable challenge for multiple enzymeꢀmediated in vitro synthesis is the tendency
for enzyme inhibition to occur by components within the complete reaction mixture.37ꢀ39 An
inspection of representative HPLC traces for the synthesis of 17a suggests this is indeed likely
the case, as several enzymatic conversions including the final Mur19ꢀcatalyzed ribosyltransfer
are incomplete (Figure 9A). As a preliminary strategy to overcome this issue and improve the
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yield, we aimed to increase the flux of the pathway by removal of the final coproduct UDP by
adding phosphoenolpyruvate and pyruvate kinase, thus enzymatically converting UDP to UTP.40
This strategy is also appealing as it would concurrently provide more starting reagent.
Unfortunately, however, no improvement was observed. Nevertheless, with the assays for each
enzyme now in hand, the potential kinetic liabilities for every enzyme catalyst—along with the
many documented factors that can contribute to the flux—can now be closely examined to
optimize the yield.
10
11
12
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CONCLUSION
The biosynthetic mechanism leading to the ADRꢀGlyU disaccharide core of 7ꢀ9 has now been
defined (Figure 10). Six enzymes were functionally assigned and characterized: Mur16, a nonꢀ
heme, Fe(II)ꢀdependent αKG:10 dioxygenase; Mur17, a PLPꢀdependent
Mur20, an ꢀTyr:11 aminotransferase stimulated by PLP; Mur26, a 13 phosphorylase; Mur18, a
UTP:5ꢀaminoꢀ5ꢀdeoxyꢀαꢀ ꢀriboseꢀ1ꢀphosphate uridylyltransferase; and Mur19, a 15:12
LꢀThr:11a transaldolase;
L
D
ribosyltransferase. Several discoveries were uncovered that are consistent with the structural
variations of 7ꢀ9 in comparison to 1ꢀ6. Notably Mur16, in contrast to the ortholog LipL involved
in 4 biosynthesis, is able to catalyze the hydroxylation of 10b along with 10a, thereby initiating
the biosynthesis of the 2ꢀdeoxyꢀADRꢀcontaining muraymycins exemplified by 9. Mur20, with
37% sequence identity with LipO, catalyzes transamination of 11 like LipO yet prefers
LꢀTyr
instead of ꢀMet as the amine donor. Following the Mur20ꢀcatalyzed reaction, the phosphorylase
L
Mur26 and nucleotidylyltransferase Mur18 work in tandem to generate an activated sugar.
Mur26 initiates this activation by generating the riboseꢀ1ꢀphosphate. Importantly, Mur26 cannot
utilize the methyletherꢀcontaining 13c, thus suggesting that Oꢀmethylation—catalyzed by an
unidentified enzyme—occurs following this step. Mur18 completes the sugar activation by
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generating the UDPꢀsugar. Unlike the typical nucletodiylyltransferase, Mur18 absolutely
requires an amine functionality to form the activated sugar, thus joining LipN as a unique group
of nucleotidylyltransferase enzymes with respect to this functional group specificity. Finally, the
ribosyltransferases Mur19 and LipN were characterized with the bona fide biosynthetic pathway
intermediates for the first time to reveal that 5′S,6′Sꢀ12 is the likely in vivo sugar acceptor and Nꢀ
alkylation follows ribosylation. Based on the in vitro characterization of these recombinant
proteins, an enzymatic synthesis of 17a (or 17b) starting from 10a (or 10b) was achieved,
providing the opportunity to explore downstream enzymatic conversions, highlighted by Nꢀ
alkylation, that form the basic scaffold that is shared among several promising nucleoside
antibiotics.
10
11
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Figure 10. Biosynthesis of the ADRꢀGlyU disaccharide core of muraymycins. The results are consistent with Oꢀ
methylation occurring downstream of ADRꢀGlyU formation. αKG, alphaꢀketoglutarate; 4ꢀHPP, 4ꢀ
hydroxyphenylpyruvate.
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EXPERIMENTAL SECTION
General Experimental Methods: UV spectra were recorded on an Ultraspec 8000 spectrometer
7
8
9
1
13
(GE, Pittsburgh, PA, USA). NMR data were recorded at 400 MHz for H and 100 MHz for C
with Varian Inova NMR spectrometers (Agilent, Santa Clara, CA). HRMS spectra were recorded
on an AB SCIEX QꢀTOF 5600 System (AB Sciex, Framingham, MA, USA). Analytic HPLC
was performed with Dionex Ultimate 3000 or Agilent 1200. Semipreparative HPLC was
performed with a Waters 600 controller and pump (Milford, MA) equipped with a 996 diode
array detector, 717 plus autosampler, and an Apollo C18 column (250 × 10 mm, 5 µm) purchased
from Grace (Deerfield, IL). All solvents used were of HPLC grade and purchased from Fisher
Scientific. Muraymycins D1ꢀD4 were isolated from Streptomyces sp. NRRL 30475 as
described.29 The synthesis and analytical characterization of 11a,16 12,17,24,26 13a,18 and 13b,18
have been previously reported (Figure S24ꢀS27).
10
11
12
13
14
15
16
17
18
19
20
21
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Synthesis of 10c. The synthesis of 10c followed a previously described procedure.16 Briefly,
pyrophosphoryl chloride (0.1 mL) was added to a cooled (4 °C) solution of 2′ꢀOꢀmethyluridine
(52 mg) in mꢀcresol (2 mL). The mixture was stirred for 2 h at 4 °C, diluted with iceꢀcold water
(7 mL), and extracted with diethyl ether (3 mL). The aqueous layer was adjusted to pH = 2 with
4 M sodium hydroxide. After lyophilization, the product was purified by HPLC equipped with a
semipreparative Apollo C18 column (250 mm × 10 mm, 5 µm). A series of linear gradients was
developed from 0.025% trifluoroacetic acid in water (A) to 0.025% trifluoroacetic acid in
acetonitrile (B) in the following manner (beginning time and ending time with linear increase to
% B): 0 min, 3% B; 0ꢀ8 min, 10% B; 8ꢀ9 min, 100% B; 9ꢀ13 min, 100% B; and 13ꢀ14 min, 3%
1
B. The flow rate was kept constant at 3.5 mL/min, and elution was monitored at 260 nm. H
NMR (400 MHz, D2O) δ 7.83 (d, J = 8.1 Hz, 1H), 5.92 (d, J = 4.1 Hz, 1H), 5.83 (d, J = 8.2 Hz,
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1H), 4.35 (t, J = 5.3 Hz, 1H), 4.21 – 4.16 (m, 1H), 4.15 (q, J = 2.8 Hz, 1H), 4.11 – 4.04 (m, 1H),
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3.96 (dd, J = 5.2, 4.2 Hz, 1H), 3.43 (s, 3H). C NMR (100 MHz, D2O) δ 166.0 151.3, 141.2,
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102.2, 87.0, 82.8, 82.5, 67.8, 64.2, 58.0. HRMS (ESI/QꢀTOF) m/z: [M – H]ꢀ Calcd for
C10H14N2O9P 337.0437; Found 337.0438.
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Synthesis of 17a. The stereoselective synthesis of the disaccharide 17a followed a previously
described procedure,30 which is based on the methodology first reported by Hirano et al.4,34,35 1
H
NMR (500 MHz, D2O) δ 7.74 (d, J = 8.0 Hz, 1H), 5.87 (d, J = 8.0 Hz, 1H), 5.81 (d, J = 2.7 Hz,
1H), 5.17 (s, 1H), 4.50 (br s, 1H), 4.35 (dd, J = 5.0, 2.7 Hz, 1H), 4.18ꢀ4.26 (m, 2H), 4.08ꢀ4.17
13
(m, 3H), 3.88 (br s, 1H), 3.25ꢀ3.38 (m, 1H), 3.06ꢀ3.15 (m, 1H); C NMR (126 MHz, D2O) δ
167.4, 152.3, 141.7, 109.0, 102.0, 91.0, 84.7, 79.1, 77.2, 75.0, 73.1, 72.0, 69.3, 57.2, 42.5;
HRMS (ESI/QꢀTOF) m/z: [M + H]+ Calcd for C16H25N4O11 449.1520; Found 449.1462; IR
(ATR) ν 3139, 2935, 2365, 2336, 1703, 1684, 1627, 1510, 1470, 1395, 1272, 1197, 1121, 1051,
1005, 819, 796, 720; UV (H2O) λmax (log ε) 202 (2.13), 262 (2.10); mp 172 °C (decomposition);
20
TLC Rf 0.05ꢀ0.16 (5:2:1 iꢀPrOHꢀH2OꢀAcOH as saturated NaCl solution); [α]D +3.8 (c 0.90,
H2O).
Synthesis of 18. Protected (5′S,6′S)ꢀ12 (previously named S3) was synthesized as described.29
The synthesis of 18 starting from protected (5′S,6′S)ꢀ12 followed a previously described method
with minor modifications.36 Protected GlyU (100 mg, 0.19 mmol) and 10% Pd/C (10 mg) in
MeOH (5 mL) was vigorously stirred under H2 atmosphere at rt for 4 h. The reaction was filtered
through
a
Celite pad and dried at room temperative.
A
solution of 3ꢀ
[(benzyloxycarbonyl)amino]propionaldehyde (78 mg, 0.3 mmol) and NaBH3CN (80 ꢁL, 1.3
mmol) in THF (2 mL) was added to the product and stirred at room temperature for 3 h,
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quenched by adding 500 µL water, and dried. Without purification, the final deprotections were
carried out according to the described protocol36 by sequential treatment with LiOH, THFꢀaq,
TFAꢀaq, and Pd/C H2 to successfully afford 18. 1H NMR (400 MHz, D2O) δ 8.02 (d, J = 8.1 Hz,
1H), 5.92 (d, J = 3.7 Hz, 1H), 5.89 (d, J = 8.1 Hz, 1H), 4.38 (d, 1H), 4.32 (d, J = 5.4 Hz, 1H),
4.27 (d, J = 5.7 Hz, 1H), 4.24 (d, J = 5.7 Hz, 1H), 3.84 (d, J = 6.3 Hz, 1H), 3.22 (m, 2H), 3.10 (t,
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13
J = 7.5 Hz, 2H), 2.12 (dt, J = 7.6, 3.6 Hz, 2H). C NMR (100 MHz, D2O) δ 173.77, 169.22,
154.03, 145.40, 105.25, 93.07, 87.08, 76.11, 72.82, 70.72, 51.37, 47.34, 39.50, 26.62. HRMS
(ESI/QꢀTOF) m/z: [M + H]+ Calcd for C14H23N4O8 375.1516; Found 375.1510.
Cloning, Overexpression and Purification of Recombinant Proteins. The genes mur16, 17,
18, 19, 20 and 26 were amplified by PCR from genomic DNA extracted from Streptomyces sp.
NRRL 30473 using Phusion Hot Start II DNA Polymerase from Thermo Scientific with supplied
HF buffer and 10 mM each of the following primer pairs: mur16 (forward) 5′ꢀ
GGTATTGAGGGTCGCGTGGTCCGCGCTGAC
AGAGGAGAGTTAGAGCCTCAGGGGCTCTCCAG
GATAGGCATATGACCTCTTCGGACGACTGC
CGAGTTGGATCCTCAGCCATGGAAGAGTCCGG
ꢀ3′
ꢀ3′;
ꢀ3′
ꢀ3′;
/
(reverse)
(forward)
(reverse)
(forward)
(reverse)
5′ꢀ
5′ꢀ
5′ꢀ
5′ꢀ
5′ꢀ
mur17
/
mur18
GGTATTGAGGGTCGCATGGCTGACTTCGCCGAACC
ꢀ3′
/
AGAGGAGAGTTAGAGCCTCATGACCAGCTCCCCGGA ꢀ3′; mur19 (forward) 5′ꢀ
AAAAAACATATGAGCCGCCCGACAAGAGT
AAAAAAGGATCCTCACAGGGTCGTAGTTCTCAG
GGTATTGAGGGTCGCGTGAGCCCCCAGAGCG
ꢀ3′
ꢀ3′;
ꢀ3′
/
(reverse)
(forward)
(reverse)
5′ꢀ
5′ꢀ
5′ꢀ
mur20
/
AGAGGAGAGTTAGAGCCTCAGGCCGTCGCCTCG ꢀ3′;
GGTATTGAGGGTCGCATGAGCACCTCCCTCGCG
and mur26 (forward) 5′ꢀ
(reverse) 5′ꢀ
ꢀ3′
/
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AGAGGAGAGTTAGAGCCTCACAGGACGGAGTGCACC ꢀ3′. Purified PCR products were
inserted into pETꢀ30 Xa/LIC (Novagen) or digested with NdeI/BamHI and ligated into pXY200
following standard prodcedure. PCR integrity was confirmed by DNA sequencing (ACGT, INC).
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Plasmids pET30ꢀmur16, pET30ꢀmur18, pET30ꢀmur20 and pET30ꢀmur26 were introduced into
E. coli BL21(DE3) cells, and the transformed strains were grown in LB supplemented with 50
ꢁg/mL kanamycin. Following inoculation of 500 mL of LB with 50 ꢁg/mL kanamycin, the
cultures were grown at 37 °C until the cell density reached an OD600 ~ 0.5 when expression was
induced with 0.1 mM IPTG. Cells were harvested after an overnight incubation at 18 °C and
lysed in 100 mM KH2PO4, 300 mM NaCl, and 10 mM imidazole (pH 8.3) using a Qsonica
sonicator (Qsonica LLC, Newtown, CT) for sonication for a total of 2 min at 40% amplitude
with 2 s pulses separated by 8 s rest periods. Following centrifugation the protein was purified
using affinity chromatography with HisPurTM NiꢀNTA agarose (Thermo Scientific, Rockford,
IL), and proteins were eluted with increasing concentrations of imidazole in Buffer A. Purified
proteins were concentrated and buffer exchanged into 25 mM KH2PO4 and 100 mM NaCl (pH
8.3) using Amicon Ultra 10,000 MWCO centrifugal filter (Millipore) and stored as glycerol
stocks (40%) at ꢀ20 °C. Protein purity was assessed as by 12% acrylamide SDSꢀPAGE; His6ꢀ
tagged proteins were utilized without further modifications. Protein concentration was
determined using UV/Vis spectroscopy, and the extension coefficients were calculated using the
ProtParam tool available from ExPASY.
Plasmids pXY200ꢀmur17 and pXY200ꢀmur19 were transformed into S. lividans TK24 using
PEGꢀmediated protoplast transformation and plated on R2YE supplemented with 50 ꢁg/mL
apramycin. After 6 days at 28 °C, positive transformants were confirmed by colony PCR using
InstaGene Matrix from BioꢀRad (Hercules, CA) and LAꢀTaq polymerase with GC buffer I. The
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recombinant strains were utilized to inoculate 50 mL R2YE containing 50 ꢁg/mL apramycin,
grown for 3 days at 28 °C at 250 rpm, and 2 mL transferred to fresh 100 mL containing 50
ꢁg/mL apramycin. Following growth for 3 days at 28 °C at 250 rpm, protein expression was
induced by addition of thiostrepton (5 ꢁg/mL) and the culture was incubated for another 24 h
before harvesting. The cells from 400 mL of culture were collected by centrifugation. The pellet
was thoroughly resuspended in iceꢀcold lysis buffer and supplemented with 4 mg/mL of
lysozyme after suspension. After incubation at 30 °C for 30 min, the cell suspension was mixed
by pipetting and lysed using a Qsonica sonicator (Qsonica LLC, Newtown, CT) for sonication
for a total of 4 min at 40% amplitude with 2 s pulses separated by 8 s rest periods. The remaining
steps were performed as described above.
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Activity Assay for Mur16. Reactions consisted of 50 mM HEPES (pH 7.5), 2.5 mM αKG, 2
mM ascorbic acid, 0.2 mM FeCl2, 1 mM 10a, 10b, or 10c, and 100 nM Mur16 at 30 °C.
Reactions were terminated by ultrafiltration using Amicon® Ultra centrifugal filter units.
Following centrifugation, the filtrate was analyzed by HPLC or LCMS equipped with an
analytical Apollo C18 column (250 mm × 4.6 mm, 5 ꢀm). A series of linear gradients was
developed from 0.1% formic acid in water (C) to 0.1% formic acid in acetonitrile (D) in the
following manner (beginning time and ending time with linear increase to % D): 0 min, 1% D; 0ꢀ
16 min, 20% D; 16ꢀ28 min, 100% D; 28ꢀ37 min, 100% D; and 37ꢀ38 min, 1% D. The flow rate
was kept constant at 0.5 mL/min, and elution was monitored at 260 nm.
Kinetics of Mur16. To determine the kinetic constants, reactions were carried out in 50 mM
TrisꢀHCl (pH 7.5), 2 mM αKG, 2 mM ascorbic acid, 0.4 mM FeCl2 with variable concentration
of 10a, 10b or 10c (1 µMꢀ500 or 1000 µM), and 40 nM Mur16 at 30 °C for 4 min. Phosphate
formation was monitored using the malachite green binding assay and the formation of aldehyde
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11a, 11b or 11c was monitored by HPLC. Each data point represents three replicate end point
assays. Kinetic constants were obtained by nonlinear regression analysis using GraphPad Prism 7
(GraphPad Software, La Jolla, CA).
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Activity Assay for Mur17. Reactions consisted of 50 mM HEPES (pH 7.5), 2 mM 11a, 11b, or
11c, 5 mM LꢀThr, 0.1 mM PLP and 1 ꢁM Mur17 at 30 °C. Following removal of the protein by
ultrafiltration, the reaction was analyzed by LCMS using the conditions described for Mur16.
Alternatively, the reaction components or 12 standards were modified with 6ꢀaminoquinolylꢀNꢀ
hydroxysuccinimidyl carbamate (AQC) prior to analysis in a reaction consisting of 20 ꢁL
sample, 60 ꢁL of 0.2 M sodium borate buffer (pH 8.8) and 20 ꢁL of 3 mg/mL AQC acetonitrile
solution. Samples were incubated at 55 °C for 10 min and then cooled to room temperature. The
AQCꢀderivatized samples (50 ꢁL) were applied to LCMS equipped with an analytical AcclaimTM
120 C18 column (100 mm × 4.6 mm, 5 µm). A series of linear gradients was developed in the
following manner (beginning time and ending time with linear increase to % D): 0 min, 1% D; 0ꢀ
40 min, 20% D; 40ꢀ41 min, 100% D; 41ꢀ44 min, 100% D; and 44ꢀ45 min, 1% D. The flow rate
was kept constant at 0.4 mL/min, and elution was monitored at 260 nm.
Activity Assay of Mur20. Reactions consisted of 50 mM potassium phosphate (pH 7.5), 1 mM
11a, 11b, or 11c, 1 mM Lꢀamino acid, and 1 ꢁM Mur20 at 30 °C. Following removal of the
protein by ultrafiltration, the reaction was analyzed by LCMS using the conditions described for
Mur16 or equipped with an analytical Apollo C18 column (250 mm × 4.6 mm, 5 ꢀm). A series
of linear gradients was developed from 40 mM triethylamineꢀacetic acid (pH 6.5) in water (E) to
40 mM triethylamineꢀacetic acid (pH 6.5) in 20% methanol (F) in the following manner
(beginning time and ending time with linear increase to % F): 0 min, 0% F; 0ꢀ8 min, 100% F; 8ꢀ
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18 min, 60% F; and 18ꢀ19 min, 0% F. The flow rate was kept constant at 1.0 mL/min, and
elution was monitored at 260 nm.
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To identify the production of αꢀketoꢀγꢀmethylthiol butyric acid, reaction mixtures or the
positive control (αꢀketoꢀγꢀmethylthiol butyric acid) were treated with 3ꢀmethylꢀ2ꢀ
benzothiazolinone hydrazone hydrochloride (MBTH) according to the method described by
Tanaka.28 Following removal of the protein by ultrafiltration, the filtrate (50 ꢁL) was mixed with
50 ꢁL of 1 M sodium acetate (pH 5.0) and 50 ꢁL of 8 mM MBTH aqueous solution. The
mixtures were incubated at 50 °C for 30 min and analyzed by LCMS equipped with an analytical
AcclaimTM 120 C18 column (100 mm × 4.6 mm, 5 µm). A series of linear gradients was
developed in the following manner (beginning time and ending time with linear increase to %
D): 0 min, 10% D; 0ꢀ10 min, 40% D; 10ꢀ20 min, 100% D; 20ꢀ27 min, 100% D; and 27ꢀ28 min,
10% D. The flow rate was kept constant at 0.4 mL/min, and elution was monitored at 350 nm.
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Activity Assay of Mur26. Reactions consisted of 50 mM potassium phosphate (pH 7.5), 2 mM
13a or 13b (13c was generated in situ with Mur16 and Mur20) and 1 ꢁM Mur26 at 30 °C.
Following removal of the protein by ultrafiltration, the filtrate was analyzed by LCMS using the
conditions described for Mur16.
Activity Assay of Mur18. Reactions of Mur18 consisted of 50 mM potassium phosphate (pH
7.5), 2 mM 13a or 13b, 5 mM MgCl2, 2 mM UTP, 1 ꢁM Mur26 (or LipP) and 1 ꢁM Mur18.
Following removal of the protein by ultrafiltration, the filtrate was analyzed by LCMS using the
conditions described for Mur20 with a triethylamineꢀacetic acid mobile phase or with an
analytical Apollo C18 column (250 mm × 4.6 mm, 5 ꢀm). A series of linear gradients was
developed in the following manner (beginning time and ending time with linear increase to %
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