Enzymatic Activity of a Glycosyltransferase Encoded in the Kanamycin Biosynthetic Gene Cluster

Enzymatic activity of a glycosyltransferase encoded in the kanamycin biosynthetic gene

cluster

Fumitaka Kudo, Hilda Sucipto, Tadashi Eguchi*

Department of Chemistry, Tokyo Institute of Technology, and Department of Chemistry and

Materials Science, Tokyo Institute of Technology, 2-12-2 O-okayama, Meguro-ku, Tokyo 152-8551

Supplementary information

Cloning of the kanM2 gene

Streptomyces kanamyceticus JCM 4433 was used as a source of chromosomal DNA to clone the kanM2 gene. General DNA manipulations in Escherichia coli were performed according to the standard protocol. E. coli DH5a was used as a host strain for the routine sub-cloning of DNA fragments. E. coli BL21(DE3) was used for expression of the kanM2 gene. The pT7-blue T vector

(Takara Bio Inc., Otsu, Shiga, Japan) was routinely used as a plasmid vector for the sub-cloning of

PCR amplified DNA. E. coli containing plasmid were cultured overnight in Luria-Bertani (LB) medium with 100 mg/ml of ampicillin or 30 mg/ml of kanamycin at 37°C to obtain plasmids, which were purified with QIAprep Spin Miniprep Kit (Qiagen K.K., Tokyo, Japan). Oligonucleotides were purchased from FASMAC (Kanagawa, Japan). Restriction enzymes and modification enzymes were purchased from Takara. PCRs were performed by GeneAmp PCR System 9700 (PE

Applied Biosystems, CA, USA) using PrimeSTAR HS polymerase (Takara). DNA sequence analysis was carried out with a LONG READER 4200 (Li-Cor, Lincoln, NE, USA) and a

SequiTherm EXCELTM II DNA Sequencing Kit (Epicentre, Madison, WI, USA) according to the

1 manufacturer’s protocol.

The kanM2 gene was amplified by PCR with primers kanM2-N: 5’-

GCATATGACCGAGCCTGCCAAGGGTG-3’ and kanM2-C: 5’-

CGGGGTGGGGGGACTCGAGGAGGACC-3’. PCR conditions were 30 cycles of 98 °C, 10 sec,

55 or 58 °C, 5 sec, 72 °C, 120 sec for extension of DNA in 2 ml of 5 x PrimeSTAR buffer, 0.8 ml of dNTP (2.5 mM each), 0.5 ml of DMSO, 0.3 ml of primer-N (10 mM), 0.3 ml of primer-C (10 mM),

0.1 ml of PrimeSTAR polymerase (Takara), 0.5 ml of genomic DNA (9 ng/ml), and 5.5 ml of water.

The amplified PCR product was treated with Ex Taq polymerase (Takara) and dNTP to attach A at the end of the fragments (72 °C, 2 min) and the resulting DNA fragments were sub-cloned into the

T vector of pT7Blue to obtain pT7Blue-kanM2. After confirmation of the DNA sequence, an appropriate plasmid was digested with NdeI and XhoI and the resulting DNA fragment was inserted into the corresponding site of pET28a (Novagen, Darmstadt, Germany) to obtain pET28-kanM2, which was then introduced into E. coli BL21(DE3) by a standard chemical transformation for overexpression.

Expression of KanM2

E. coli BL21(DE3) harboring pET28-kanM2 was inoculated into 3 mL of LB medium with 30

μg/ml kanamycin. After incubation for 15 h at 37°C, 2-3 mL of the pre-culture was used to

inoculate 200 mL of LB medium. Bacteria were cultured at 37°C until OD600 reached 0.6-1.0. IPTG was added to a final concentration of 0.5 mM for induction, and cultures were continued for an additional 19 h at 15°C. The cells were harvested by centrifugation at 8,000 rpm for 30 min, and the resulting cells were stored at -30°C for further use. One gram of cells was suspended in 10 mL buffer and cell-free extracts were prepared by sonic oscillation (Sonifier Type-250, Branson,

Danbury, CT, USA, 1 min x 5 times, output 2, duty cycle 50%). The resulting cell lysate was

2 separated by centrifugation at 7,000 rpm for 30 min at 4°C. The supernatants were used as cell-free extracts (CFE) containing the expressed protein. The obtained proteins were confirmed by a standard SDS-PAGE analysis.

Enzyme assays with recombinant KanM2 protein

Optimum buffer conditions for the KanM2 reaction were 50 mM Tris buffer or HEPES buffer (pH

8.0) containing 10% glycerol, 1 mM MgCl2. The expressed protein in CFEs was mixed with 2 mM of sugar acceptor (20 mM, 2 μl of paromamine) and 2 mM of glycosyl donors (20 mM, 2 μl of

UDP-Glc; UDP-GlcNAc) to total volume 20 μl, and the solutions were incubated at 28°C for 18 h.

The enzymatic reactions were stopped by addition of 20 μl ethanol. After centrifugation, an aliquot

(20 μl) of the enzymatic solution was taken, and treated with 10 μl of DMSO, 20 μl of 5% solution of 2,4-dinitrofluorobenzene (DNFB) in methanol, and 2 μl of 2 M NaOH at 60°C for 1.5 h. After that, 50 μl of water was added, then dinitrophenyl derivatives were extracted with 500 μl ethyl acetate. The solvent of the ethyl acetate layer was removed by a centrifugal evaporator. The residues were dissolved in 100 μl methanol for HPLC and LC-ESI-MS analysis.

HPLC analysis was performed on a pump (L-7100, Hitachi, Japan) equipped with a column oven (L-7300, Hitachi), with PEGASIL ODS (4.6 x 250 mm, Senshu Pak, Japan), a UV detector (L-7405, Hitachi), and a Chromato-Integrator (D-2500, Hitachi). A mobile phase system consists of Milli-Q water and methanol was used with the following gradient: 0 – 30 min a linear gradient from 50% MeOH to 90% MeOH, 31 – 35 min isocratic at 100% MeOH. Each sample (5

μl) was injected into the HPLC. Flow rate used was 0.9 mL/min. Column oven was 40°C and UV absorption at wavelength 350 nm was recorded.

LC-ESI-MS analysis was performed using a mass spectrometer (LCQ; Finnigan, Thermo

Fisher Scientific, Waltham, MA, U.S.A.) coupled with HPLC (Nanospace SI-1; Shiseido Co. Ltd.,

3 Tokyo, Japan). An aliquot of the solution (2 l) after derivatization was injected into the LC system equipped with a Mightysil RP-18 GP 100-1.0 (3 m; Kanto Chemical Co. Inc., Tokyo, Japan).

Elution was done using 10% aqueous CH3CN for 5 min, followed by a linear gradient of 50–80%

aqueous CH3CN for 40 min at a flow rate of 50 μL/min, 40°C. Elution was monitored at 350 nm.

The ESI mass spectrometer was operated in the negative-ion mode.

Isolation of KanM2 reaction product, 3”-deamino-3”-hydroxykanamycin C

Two grams of the wet cells containing KanM2 were suspended in 20 mL HEPES buffer (50 mM,

pH 8.0), 1 mM MgCl2, 10% glycerol. The suspension was then disrupted by sonic oscillation (10 x

1 min), output 2, duty cycle 50% and were centrifuged at 10,000 rpm for 30 min, 4°C. The cell-free extract was subjected to ammonium sulfate fractionation at 10% and 40% saturation using solid

(NH4)2SO4. Fractionation was carried out by stirring for 50 min at 4°C. Every precipitate in each saturation range was collected by centrifugation at 4°C for 15 min at 10,000 rpm. The precipitates were suspended in 1.5 mL of the above-mentioned buffer.

The large-scale enzyme reaction was carried out at 28°C with 10-40% (NH4)2SO4 fractionated enzyme solution (1 mL), paromamine (3 mM, 10 mg), and UDP-Glc (4.7 mM, 29 mg)

in 50 mM HEPES buffer (pH 8.0) containing 1 mM MgCl2 and 10% glycerol (total 10 mL). After

30 h, another 5 mg of UDP-Glc and 0.25 μl of enzyme solution were added. After another 25 h, another 5 mg of UDP-Glc and 0.25 μl of enzyme solution were added. The total enzyme reaction time was 92 h. To consume the remained paromamine, the second enzyme reaction was carried out with the same procedure as stated above using 1 g of fresh cells and 18.9 mg of UDP-Glc. 18 mg of

UDP-Glc and enzyme were added everyday, for 2 times (96 h). Furthermore, the third enzyme reaction was also carried out for 2 days with the same method as the first and the second enzyme reaction. After every addition of enzyme and UDP-Glc, the conversion ratio was estimated by

4 taking 10 μl aliquot from the reaction solution. The aliquot was derived to dinitrophenyl derivative and then analyzed by HPLC.

The enzymatic solution was acidified with acetic acid to pH 5.0. The acidified solution

+ was then applied to Amberlite CG50 (Rohm and Haas Co, Philadelphia, PA, U.S.A., NH4 form, 0.5 ×

12 cm), washed with water (50 mL) followed by 0.1 mM aqueous NH4OH (100 mL), then the

expected product was eluted with 1.0 M aqueous NH4OH (100 mL). Each eluted fraction was 8

mL. Every fraction was checked by TLC through developing solvent CHCl3 : MeOH : NH4OH :

EtOH = 4 : 6 : 7 : 1. The solvent of the product containing fractions was removed by a rotary evaporator and the concentrate was applied to a DOWEX AG1-X8 (Bio-Rad, Richmond, CA,

2- U.S.A., SO4 form, 0.5 × 10 cm) column to obtain aminoglycoside sulfate (7.9 mg). The NMR data were recorded (DRX500; Bruker BioSpin, Rheinstetten, Germany, or ECX400; JEOL, Akishima,

Tokyo, Japan) in deuterium oxide (99.8% atom D; Acros Organics, Ceel, Belgium). 1H NMR (500

MHz, D2O, Figure S1): δ 1.67 (q, J = 12.6 Hz, 1H, H-2ax), 2.32 (dt, J = 12.6 and 4.0 Hz, 1H, H-

2eq), 3.28 (dd and m, J = 3.9 and 10.7 Hz, 2H, H-2’ and H-1 or H-3), 3.36 (t, J = 9.4 Hz, 1H), 3.40

(t and m, J = 9.4 Hz, 2H), 3.55–3.85 (m, 12H), 4.99 (d, J = 3.7 Hz, 1H, H-1”), 5.52 (d, J = 3.9 Hz,

13 1H, H-1’); C NMR (125 MHz, D2O, 1,4-dioxane δ 66.5 was used as an internal standard. Figure

S2): δ 29.6 (C-2), 48.7 (C-1 or C-3), 49.9 (C-1 or C-3), 54.2 (C-2’), 60.3 (C-6’ or C-6”), 60.4 (C-6’ or C-6”), 69.2, 69.4, 69.6, 71.5, 72.7, 72.8, 73.4, 73.8 (C-5), 82.4 (C-4), 84.2 (C-6), 97.3 (C-1’),

101.3 (C-1”). Therein, x indicates signals for remaining paromamine and other impurities. 2D-

NMRs including 1H-1H COSY, HMQC, HMBC and TOCSY (mixing time 30 and 150 ms) spectra were also recorded on DRX500 or ECX400 to determine the structure (data not shown). The key

NMR correlations for structure determination are shown in Figure 3S. HR-FAB-MS (JMS700,

JEOL, Akishima, Tokyo, Japan), (positive mode, glycerol): m/z [M+H]+ 486.2307, calculated for

C18H36O12N3: 486.2299.

5 Figure legends for the Supporting information

Figure S1

1 H NMR of 3”-deamino-3”-hydroxykanamycin C (500 MHz, D2O)

Figure S2

13 C NMR of 3”-deamino-3”-hydroxykanamycin C (125 MHz, D2O)

Figure S3

The key NMR correlation information leading to the structure elucidation

Bold lines are determined from 1H-1H COSY and the arrows show the HMBC correlation.

6 Figure S1

7 Figure S2

8 Figure S3

OH HO O HO 1' NH2 2 H2N O 4 HO 6 NH2 O OH 1" O HO OH OH 1H-1H COSY HMBC