Characterization of protein encoded by spnR from the spinosyn gene cluster of Saccharopolyspora spinosa: Mechanistic implications for forosamine biosynthesis

Article abstract of DOI:10.1021/ja042702k

d-Forosamine is a 4-N,N-(dimethylamino)-2,3,4,6-tetradeoxy-α-d-threo-hexopyranose found in spinosyn produced by Saccharopolyspora spinosa. Studies of spinosyn biosynthesis in S. spinosa led to the isolation of the entire biosynthetic gene cluster. Heterol

Full text of DOI:10.1021/ja042702k

Published on Web 05/06/2005
Characterization of Protein Encoded by spnR from the Spinosyn Gene
Cluster of Saccharopolyspora spinosa: Mechanistic Implications for
Forosamine Biosynthesis
Zongbao Zhao, Lin Hong, and Hung-wen Liu*
DiVision of Medicinal Chemistry, College of Pharmacy, and Department of Chemistry and Biochemistry,
UniVersity of Texas, Austin, Texas 78712
Received December 4, 2004; E-mail: h.w.liu@mail.utexas.edu
Scheme 1
Deoxysugars are constituents of many biologically active natural
products in which they are commonly the determinants of activity
for the parent molecules.1 To characterize deoxysugar biosynthetic
pathways and to collect genes useful for in vivo glycodiversification
of secondary metabolites,2 we investigated the biosynthesis of
D-forosamine (1, 4-N,N-(dimethylamino)-2,3,4,6-tetradeoxy-R-D-
threo-hexopyranose). This unusual sugar is found in several natural
products, including spiramycin,3 a clinically useful antibiotic, and
spinosyn (2),4 an environmentally benign insecticide. The structure
of forosamine is unique due to its highly deoxygenated nature, and
its biosynthesis must include a series of C-O bond cleavage events.
Early studies of spinosyn production in Saccharopolyspora spinosa
led to the isolation of the entire biosynthetic gene cluster, in which
spnN, spnO, spnP, spnQ, spnR, and spnS, were proposed to be
involved in forosamine formation.5 The tentative identification of
these genes by sequence analysis allowed the assignment of
potential roles for the gene products, from which two possible routes
starting from 3 can be envisioned for the biosynthesis of 1 (Scheme
1). The key steps in route A involve deoxygenation at C-2 (3 f 4)
and then at C-3 (4 f 6), followed by transamination at C-4 (6 f
7) and dimethylation of the 4-amino group to give TDP-forosamine
(8), which is then incorporated into 2. The reaction sequence of
route B closely resembles that of route A except for the order of
the C-3 deoxygenation and C-4 transamination steps (4 f 9 f 7),
both of which are expected to be catalyzed by B6-dependent
enzymes.
The C-3 deoxygenation step is of particular interest since this
transformation in the formation of other glucose-derived 3,6-
dideoxysugars (such as ascarylose) requires a pair of enzymes, a
B6-dependent [2Fe-2S]-containing dehydrase (E1), and an iron-
sulfur flavoprotein reductase (E3), acting in concert.6,7 The typical
As shown in Scheme 1, the substrate and the cofactor require-
ments for the C-3 deoxygenation by SpnQ and C-4 transamination
by SpnR are clearly different depending on which route is used
during forosamine production. Hence, the sequence of events in
this pathway can be established by identifying the function of SpnQ.
However, difficulties were encountered in our attempts to express
the spnQ gene to directly examine the catalytic properties of purified
SpnQ. To circumvent these obstacles, we investigated the substrate
specificity of SpnR, using a stable TMP-phosphonate analogue of
7. Our characterization of SpnR identifies it as an aminotransferase
that catalyzes the interconversion between 6 and 7. This result
indicates that route A is preferred in the forosamine biosynthetic
pathway.
reaction, illustrated in route A, proceeds via a dehydration/electron-
transfer reduction mechanism (5a f 5b f 5c). Sequence identity
(49%) between SpnQ, the gene product of spnQ, and E1 of the
ascarylose pathway8 implicates SpnQ as the likely E1-equivalent
that converts 5a f 5b in the forosamine biosynthesis. The
conservation of an “E1-type” [2Fe-2S] binding motif9 in SpnQ
further suggests that an electron-transfer reduction is an integral
part of the deoxygenation reaction in which the [2Fe-2S] center
in SpnQ serves as a part of the electron-transfer conduit. However,
no E3 equivalent gene was found in the spn gene cluster. A generic
reductase could function as an E3 surrogate in the conversion of 4
to 6 (route A, 5b f 5c), following the mechanism established for
ascarylose and other glucose-derived 3,6-dideoxysugars. Alterna-
tively, the absence of an E3 equivalent gene in the spn gene cluster
might indicate that C-3 deoxygenation is not a typical E1/E3 reaction.
It may instead be accomplished by the catalysis of SpnQ along
with a nonspecific reductase on the aminosugar intermediate 9 to
give 7 (route B, 5a f 5b f 5d).
To determine the function of SpnR, the spnR gene was amplified
by the polymerase chain reaction (PCR) using genomic DNA from
S. spinosa (NRRL18537) as template and cloned into a pET24b-
(+) vector. The resulting construct, pKZR1, was used to transform
the Escherichia coli BL21(DE3) cells. The expressed SpnR,
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J. AM. CHEM. SOC. 2005, 127, 7692-7693
10.1021/ja042702k CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
Scheme 2
These results provide the first direct evidence establishing the
relevance of the spnR gene to forosamine biosynthesis. The
verification of the catalytic function of SpnR has substantiated its
assigned role as an aminotransferase in the proposed biosynthetic
pathway. Although a full understanding of the mechanism of the
C-3 deoxygenation awaits future characterization of the SpnQ
protein, our data shed light on the reaction sequence of the
forosamine pathway. In particular, the substrate for SpnQ can be
implicated as compound 4 on the basis of our results. Moreover,
the strategy of utilizing stable TMP-phosphonate sugars in place
of TDP-sugar derivatives to determine the possible functions of
proteins involved in unusual sugar biosynthesis may also find
general applicability for the design of probes to study related
enzymes whose mechanisms involve the transformation of labile
deoxysugar intermediates.
Acknowledgment. This work was supported in part by the
National Institutes of Health Grants GM35906 and 40541. We thank
Dr. K. Madduri of Dow AgroSciences for helpful discussion on
the genetics of S. spinosa.
Scheme 3
Supporting Information Available: Synthesis of 10 and spectral
data of 10 and 11 (PDF). This material is available free of charge via
the Internet at http://pubs.acs.org.
References
(1) (a) Weymouth-Wilson, A. C. Nat. Prod. Rep. 1997, 14, 99-110. (b)
Thorson, J. S.; Vogt, T. In Carbohydrate Based Drug DiscoVery: Wong,
C.-H, Ed.; Wiley-VCH: Weinheim, 2003; pp 685-711. (c) He, X.; Liu,
H.-w. Annu. ReV. Biochem. 2002, 71, 701-754.
containing a C-terminal His6-tag, was purified to near homogeneity
by a protocol consisting of Ni-NTA affinity and Sephacryl S-200
size-exclusion chromatography. The isolated SpnR is a homodimeric
protein and exhibits weak absorption at 325 and 410 nm.10
The predicted substrate for SpnR in route B is 4, which could
be prepared from 3 by the action of SpnO and SpnN in the presence
of NADPH.11 Preparation of the predicted substrate 6 for SpnR in
route A was more challenging due to the difficulties in reconstituting
SpnQ activity. Many attempts to chemically synthesize the desired
compound failed due to the facile loss of the TDP substituent at
C-1, a well-known property of 2-deoxyhexoses. Hence, instead of
6, an isostere of 7 (compound 10) in which the TMP group is joined
to the hexose core through a stable C-glycosidic phosphonate linker
was synthesized (Scheme 2).12 Because enzyme-catalyzed trans-
amination is a reversible process, if SpnR is the desired aminotrans-
ferase using 6 as the substrate, compound 10 could be converted
to the corresponding keto-sugar 11 by SpnR (Scheme 3).
(2) (a) Zhao, L.; Sherman, D. H.; Liu, H.-w. J. Am. Chem. Soc. 1998, 120,
10256-10257. (b) Borisova, S. A.; Zhao, L.; Sherman, D. H.; Liu, H.-w.
Org. Lett. 1999, 1, 133-136. (c) Zhao, L.; Ahlert, J.; Xue, Y.; Thorson,
J. S.; Sherman, D. H.; Liu, H.-w. J. Am. Chem. Soc. 1999, 121, 9881-
9882. (d) Yamase, H.; Zhao, L.; Liu, H.-w. J. Am. Chem. Soc. 2000, 122,
12397-12398. (e) Melanc¸on, C. E., III; Takahashi, H.; Liu, H.-w. J. Am.
Chem. Soc., 2004, 126, 16726-16727.
(3) (a) Matsui, D. Clin. Perinatol. 1994, 21, 675-689. (b) Poulsen, S. M.;
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2002, 95, 640-650.
(5) Waldron, C.; Matsushima, P.; Rosteck, P. R., Jr.; Broughton, M. C.; Turner,
J.; Madduri, K.; Crawford, K. P.; Merlo, D. J.; Baltz, R. H. Chem. Biol.
2001, 8, 487-499.
(6) (a) He, X.; Agnihotri, G.; Liu, H.-w. Chem. ReV. 2000, 100, 4615-4661.
(b) He, X.; Liu, H.-w. Cur. Opin. Chem. Biol. 2002, 6, 590-597.
(7) C-3 deoxygenation catalyzed by ColD in colitose biosynthesis is an
exception in which the cofactor (PMP) is regenerated by a second function
of ColD, an aminotransferring activity, instead of by the E3-catalyzed
reduction (Beyer, N.; Alam, J.; Hallis, T. M.; Guo, Z.; Liu, H.-w. J. Am.
Chem. Soc. 2003, 125, 5584-5585).
To determine the function and substrate specificity of SpnR, a
mixture of SpnR (10 µM), 10 (1 mM), R-ketoglutarate (30 mM),
and PLP (40 µM) in 100 mM potassium phosphate buffer (pH 7.5)
was incubated at 24 °C. Aliquots of the incubation mixture were
withdrawn at different time intervals from which a new product
was detected by HPLC using an anion exchange Dionex column
(4 × 250 mm).13 A large-scale preparation was then carried out,
(8) Thorson, J. S.; Lo, S. F.; Ploux, O.; He, X.; Liu, H.-w. J. Bacteriol. 1994,
176, 5483-5493.
(9) E1 and those believed to function as dehydrases have a catalytic histidine
at position 220 (using the E1 numbering system) and display an unusual,
albeit well-defined iron-sulfur binding motif. Both traits are conserved
in SpnQ sequence: Agnihotri, G.; Liu, Y.-n.; Paschal, B. M.; Liu, H.-w.
Biochemistry 2004, 43, 14265-14274.
(10) These results suggest that isolated SpnR is predominantly an apoenzyme.
Thus, PLP was included in all incubations with SpnR.
(11) Expression, purification, and characterization of SpnO and SpnN will be
reported elsewhere. The spectral data of 4 are identical to those reported
in the literature: Draeger, G.; Park, S.-H.; Floss, H. G. J. Am. Chem.
Soc. 1999, 121, 2611-2612.
1
and the isolated product was identified as 11 on the basis of H
and 13C NMR analyses.12 The conversion of 11 to 10 was also
established by HPLC.14 These results clearly demonstrated that 11/
10, and by inference 6/7, are the substrates for SpnR. Interestingly,
when the incubation was carried out using 4 as the substrate,
formation of 9 was also detected.12 However, the efficiency of 11
to 10 was much greater (more than 15-fold) than that of 4 to 9 by
SpnR under the same conditions.15 Thus, while both 4 and 11, the
isostere of 6, could be recognized and processed by SpnR, the higher
efficiency of conversion observed for 11 f 10 as compared to that
for 4 f 9 suggested that 6 is more likely the physiological substrate
for SpnR. Thus, SpnR can be referred to as TDP-4-keto-2,3,6-
trideoxy-D-glucose 4-aminotransferase.
(12) See Supporting Information for details.
(13) Baseline separation of the product 11 from the substrate 10 (retention
times ) 31.0 and 6.7 min, respectively) was achieved using a linear
gradient from 25 to 300 mM ammonium acetate (pH 7.0) over 35 min
with a flow rate of 1 mL/min.
(14) A kcat of 0.25 s-1 and a Km of 100 µM for 11 were estimated for this
reaction at room temperature. The assay mixture (100 µL) contained 1.0
µM SpnR, 250 µM PLP, 50 mM L-glutamate, 150 µM NADPH, 80 mM
NH4Cl, 0.6 U of L-glutamic dehydrogenase, and varied amounts of 11 in
50 mM potassium phosphate buffer, pH 7.5.
(15) Comparison was based on the extent of conversion measured by HPLC
in each case. Determination of kcat and Km for 4 f 9 was hampered by
the low conversion of this reaction.
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