Biosynthesis of aminoglycoside antibiotics: Cloning, expression and characterisation of an aminotransferase involved in the pathway to 2-deoxystreptamine

Article abstract of DOI:10.1039/b209799k

The gene btrR from Bacillus circulans has been cloned and expressed and shown to produce a protein which catalyses the transamination of 2-deoxy-scyllo-inosose to give 2-deoxy-scyllo-inosamine, an intermediate in the biosynthesis of 2-deoxystreptamine.

Full text of DOI:10.1039/b209799k

Biosynthesis of aminoglycoside antibiotics: cloning, expression and
characterisation of an aminotransferase involved in the pathway to
2-deoxystreptamine†
Fanglu Huang, Yanyan Li, Jinquan Yu and Jonathan B. Spencer*
Cambridge Centre for Molecular Recognition, Department of Chemistry, University of Cambridge, Lensfield
Road, Cambridge, UK CB2 1EW. E-mail: jbs20@cus.cam.ac.uk; Fax: 44 1223 336710; Tel: 44 1223
336362
Received (in Cambridge, UK) 8th October 2002, Accepted 21st October 2002
First published as an Advance Article on the web 5th November 2002
The gene btrR from Bacillus circulans has been cloned and
expressed and shown to produce a protein which catalyses
the transamination of 2-deoxy-scyllo-inosose to give
2-deoxy-scyllo-inosamine, an intermediate in the biosynthe-
sis of 2-deoxystreptamine.
In order to elucidate the complete pathway to 2-deox-
ystreptamine we have carried out further gene walking
experiments using genomic DNA from B. circulans NRRL
B3312 in an attempt to obtain the whole butirosin gene cluster.
During these studies we identified a 1257 bp open reading frame
now named btrR (accession number AJ494863, EMBL nucleo-
tide sequence database) that has homology to the aminotransfer-
ase family of enzymes. The protein sequence of BtrR contains
a highly conserved motif that has been observed in a new class
of pyridoxal 5A-phosphate (PLP)-dependent transaminase found
mainly in the biosynthesis of secondary metabolites. Two of the
transaminases involved in biosynthesis of streptamine: StsC,
Aminoglycosides are a large class of clinically important
antibiotics. They are classified into two groups depending on
the structure of the aglycone. One class, typified by streptomy-
cin and fortimicin, has the fully substituted aminocyclitol
streptamine. The other class has 2-deoxystreptamine as the
common aglycone and contains many of the clinically important
aminoglycosides like kanamycin, neomycin, gentamicin and
butirosin (Fig. 1).1
the -glutamine:scyllo-inosose aminotransferase, and StsA, the
L
L
-alanine:N-amidino-3-keto-scyllo-inosamine aminotransferase
are both members of this new class of transaminase.10
Alignment of BtrR with StsC reveals high homology (40.3%
sequence identity) between these two proteins, suggesting they
may have a similar function.
To determine whether the protein encoded by btrR catalyses
the transamination of 2 to give 2-deoxy-scyllo-inosamine (3) or
one of the other transamination steps in the biosynthesis of
butirosin, the gene was sub-cloned into a pET28 vector and the
protein expressed in E. coli BL21 (DE3). The over-expressed
His6-tagged protein was purified by Ni2+-NTA affinity column
followed by gel filtration and anion-exchange chromatography.
Purified BtrR gave a single band on SDS-PAGE gel with a
molecular mass around 50 kDa (Fig. 2). The molecular weight
of BtrR determined by LC-ESI-MS is 50 714 Da (see
supplementary information†) in agreement with the calculated
MW of 50 703 Da. Gel filtration chromatography showed an
apparent MW of 97 kDa for BtrR determining that it is a
homodimer.
Purified BtrR showed two UV-Vis absorptions, one at 335
nm and the other at 415 nm indicating the presence of BtrR-
bound pyridoxamine 5A-phosphate (PMP, 335 nm) and PLP
(415 nm), respectively. This is similar to that of StsC, one of the
transaminases in streptamine biosynthesis pathway from Strep-
tomyces griseus and is typical of PLP-containing enzymes.10
Addition of 2 significantly enhanced the absorption at 415 nm
but reduced the absorption at 335 nm suggesting amino transfer
from PMP to 2 whilst addition of 3 showed the opposite effect
Fig. 1 Structures for butirosin A and butirosin B.
Labelling studies have demonstrated that the biosynthesis of
streptamine and 2-deoxystreptamine are quite different. Strepta-
mine is formed from myo-inositol2 whereas 2-deoxystreptamine
is biosynthesised from glucose-6-phosphate (1) (Scheme 1).3–7
The initial step in the formation of 2-deoxystreptamine (5) has
been shown to be catalysed by 2-deoxy-scyllo-inosose synthase.
This enzyme converts 1 into 2-deoxy-scyllo-inosose (2) by a
mechanism analogous to that of dehydroquinate synthase.8 It
has been purified to homogeneity and the protein sequence was
used successfully to locate the gene (btrC) in the butirosin
producing organism Bacillus circulans. Subsequent gene walk-
ing from both sides of btrC identified additional genes in the
butirosin cluster.9
Scheme 1 Proposed biosynthetic pathway of 2-deoxystreptamine. 1. D-
glucose-6A-phosphate; 2. 2-deoxy-scyllo-inosose; 3. 2-deoxy-scyllo-in-
osamine; 4. 2-deoxy-3-amino-scyllo-inosose; 5. 2-deoxystreptamine.
Fig. 2 Expression of BtrR. Lane 1: before induction; lane 2: after induction;
lane 3: cell-free extract; lane 4: cell pellet; lane 5: unbound proteins; lane 6
and 7: column-wash fractions; lane 8: purified BtrR; lane 9: protein
marker.
† Electronic supplementary information (ESI) available: LC-MS, TLC and
ESI-Q-TOF MS. See http://www.rsc.org/suppdata/cc/b2/b209799k/
2860
CHEM. COMMUN., 2002, 2860–2861
This journal is © The Royal Society of Chemistry 2002

(Fig. 3).11 This clearly indicates that BtrR binds 2 and 3, thus
suggesting that 2 and 3 are the true substrates for this
enzyme.
amino acids could be taken as poor substrates by BtrR (all of the
other common amino acids were not substrates for BtrR).
BtrR was also shown to catalyse the reverse reaction when
incubated with 3 and pyruvic acid as an amino acceptor. We did
To test the activity of BtrR we carried out a series of enzymic
reactions using 2 and different amino acids as amino donors.
Each incubation was carried out for 1 h at 37 °C and contained
0.1 mg ml21 BtrR, 4 mM of 2, 5 mM of an amino acid in 30 mM
Tris·HCl buffer (pH 7.5) containing 0.3 mM PLP. TLC of the
reaction mixtures showed that a new compound with the same
not use a-keto-glutaramate (the transaminated -glutamine) due
L
to its instability in solution (cyclises spontaneously to form
5-hydroxy-2-pyrrolidone-5-carboxylic acid).10
To reconstitute the early part of the 2-deoxystreptamine
pathway we also cloned and expressed the first enzyme BtrC.
BtrC was cloned by PCR based on the known sequence,9
inserted into a pET28 vector, expressed in E. coli BL 21 (DE3)
and purified as described for BtrR except a Co2+-NTA affinity
column was used. The BtrC/BtrR coupled assay was carried out
using 1 (5 mM) as a substrate in the presence of 5 mM b-NAD,
Rf value as 3, was formed in the incubation that had -glutamine
L
as the amino donor (Fig. 4A).
After the completion of the reaction the product was purified
and determined to be 3 by ESI-Q-TOF tandem mass spectrome-
try (identical mass spectra to authentic 3, see supplementary
information†). In the reactions using
L
-alanine,
L
-serine, or
L
-
0.2 mM CoCl2, 20 mM
-glutamine, and 0.1 mg ml21 of each
L
glutamate as amino donors, formation of 3 was also observed
but only after overnight incubation at 37 °C indicating that these
of the enzymes. Analysis by TLC showed that 3 was rapidly
formed thus demonstrating that these two enzyme can act
concomitantly (Fig. 4B).
The remaining pathway to 2-deoxystreptamine is proposed to
involve the oxidation of 3 to 4, probably catalysed by a
dehydrogenase, and finally a transamination to form 5.
Interestingly, BtrR also catalysed amino transfer from 5 to
pyruvic acid and therefore it is possible that BtrR also catalyses
the transamination from 4 to 5 (the lack of a synthetic standard
of 4 has prevented us, at this stage, from confirming this
proposal).
In summary, we have cloned, expressed and characterised for
the first time -glutamine:2-deoxy-scyllo-inosose aminotrans-
L
ferase, BtrR, involved in the biosynthesis of 2-deoxystrepta-
mine from the butirosin producing strain Bacillus circulans
NRRL B3312. BtrR has been shown to exist as a homodimer
with a subunit molecular weight of 50.7 kDa. -glutamine has
L
been determined to be the best amino donor for the formation of
3 catalysed by BtrR. We have also shown that the early part of
the 2-deoxystreptamine pathway can be readily reconstituted in
vitro using recombinant BtrC and BtrR.
We are grateful to Dr Oliver W. Choroba, Tsung-lin Li and
Dr Hui Hong for MS analysis, to Joseph Wright for help in the
synthesis of 2-deoxy-scyllo-inosose and 2-deoxy-scyllo-in-
ososamine. This work is financially supported by BBSRC.
Fig. 3 UV-Vis absorption spectra of BtrR. A: with or without 2 (2-deoxy-
scyllo-inosose); B: with or without 3 (2-deoxy-scyllo-inosamine).
Notes and references
1 K. L. Rinehart, Jr. and R. M. Stroshane, J. Antibiot., 1976, 29, 319.
2 J. Distler, K. Mansouri, G. Mayer, M. Stockmann and W. Piepersberg,
Gene, 1992, 115, 105.
3 T. Furumai, K. Takeda, A. Kinumaki, Y. Ito and T. Okuda, J. Antibiot.,
1979, 32, 891.
4 N. Yamauchi and K. Kakinuma, J. Org. Chem., 1995, 60, 5614.
5 F. Kudo, N. Yamauchi, R. Suzuki and K. Kakinuma, J. Antibiot., 1997,
50, 424.
6 N. Iwase, F. Kudo, N. Yamauchi and K. Kakinuma, Biosci. Biotechnol.
Biochem., 1998, 62, 2396.
7 K. Suzukake, K. Tokunaga, H. Hayashi, M. Hori, Y. Uehara, D. Ikeda
and H. Umezawa, J. Antibiot., 1985, 38, 1211.
Fig. 4 TLC analysis of BtrR activity: 1 ml of each reaction was applied to a
silica TLC gel plate, developed in methanol:ammonia (5+1), and stained
using ninhydrin reagent. A. BtrR catalysed forward reaction. Lane I: 3
(2-deoxy-scyllo-inosamine) standard; lane II: no enzyme; lane III: no 2
(2-deoxy-scyllo-inosose); lane IV: no amino donor; from lane VI to lane X:
8 F. Kudo, H. Tamegai, T. Fujiwara, U. Tagami, K. Hirayama and K.
Kakinuma, J. Antibiot., 1999, 52, 559.
9 Y. Ota, H. Tamegai, F. Kudo, H. Kuriki, A. Koike-Takeshita, T. Eguchi
and K. Kakinuma, J. Antibiot., 2000, 53, 1158.
10 J. Ahlert, J. Distler, K. Mansouri and W. Piepersberg, Arch. Microbiol,
1997, 168, 102.
L
-glutamine, L-alanine, L-glutamate, L-serine, L-ornithine and L-arginine
were used as amino donors, respectively. B. BtrC/BtrR-coupled assay. Lane
I: no BtrR; lane II: no BtrC; lane III: BtrC + BtrR; lane IV: 3 standard.
11 2 and 3 were synthesised as detailed: J. Yu and J. B. Spencer,
Tetrahedron Lett., 2001, 42, 4219.
CHEM. COMMUN., 2002, 2860–2861
2861