ars.els-cdn.com€¦ · Web viewIdentification of the reaction product was performed through the 1H NMR and mass spectra. The 1H nuclear magnetic resonance (NMR, dimethylsulfoxide,

Discovery of a new a Fe-type nitrile hydratase efficiently hydrating

aliphatic and aromatic nitriles by genome mining

The name(s) and affiliation(s) of the author(s):

Xiaolin Pei1,2, Lirong Yang1, Gang Xu1, Qiuyan Wang2, Jianping Wu1

1 Institute of Bioengineering, Department of Chemical and Biological Engineering,

Zhejiang University, Hangzhou, 310028, PR China

2 Center for Biomedicine and Health, College of Life and Environmental Sciences,

Hangzhou Normal University, Hangzhou 310012, PR China

Correspondence:

Jianping Wu ([email protected]), Institute of Bioengineering, Department of Chemical

and Biological Engineering, Zhejiang University, Hangzhou, 310028, PR China. Tel.:

0086-571-87952363; Fax: 0086-571-87952009.

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1. Materials and Methods

1.1 Purification of the recombinant NHase_F1

For purification of the recombinant NHase_F1, the obtained supernatant was loaded

to a nickel chelate affinity column (Ni-NTA Resin, Bio Basic INC.) using loading

buffer (20 mM phosphate buffer, pH 7.5, 500 mM NaCl, and 50 mM imidazote).

Target NHase protein was recovered using elution buffer (20 mM phosphate buffer,

pH 7.5, 500 mM NaCl, and 250 mM imidazote), then desalted by HiTrap desalting

column (Amersham Biosciences).

1.2 Identification of nicotinamide

Identification of the reaction product was performed through the 1H NMR and mass

spectra. The 1H nuclear magnetic resonance (NMR, dimethylsulfoxide, 400 MHz) and

mass spectra were recorded with FX-90Q (Jeol, Japan) and LCMS-2010 (Shimadzu,

Japan) respectively.

1.3 Molecular modeling

The homology modeling of NHases from Pseudomonas putida F1 and Pseudomonas

chlororaphis B23 was conducted using Accelry Discovery Studio 3.0. The crystal

structure 3A8O, NHase from Rhodococcus erythropolis N771, was used as the model

structure. The obtained 3D structures were energy minimized using Smart Minimizer

algorithm with Max steps of 200 and RMS gradient of 0.1. The 3D structural

similarity of NHases from P. putida F1 and P. chlororaphis B23 were aligned using

Align Structures (3DMA program) with RMSD Cutoff of 2.5.

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2. Figures

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Select Conserved motifs/Blocks as Probes

Sequence Analysis and Target Selection

Sequence Library Assembly

Select target phenotype NHase protein sequencesAlign protein sequences by ClustalW2Conserved protein sequences motifs/blocks

Genome databasesGenBank

Non-redundant (nr)Search tools

BLAST

Extract and select protein sequences that contain the conserved motif

Sequence analysisAlign sequences

PhylogenyDistance

Select at least one of the gene sequences

Design One or More Degenerate Primers to Target the Selected Gene Sequences

Cloning, Expression and Characterization

Fig. S1. Methodology for Fe-type NHases by genome mining.

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Fig. S2. (A) Multiple sequence alignment of putative active site from different

NHases. (B) The crystal structure of NHase and putative active site.

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Fig. S3. SDS-PAGE of purified recombinant NHase_F1 fractions. M: molecular

weight standard, lane 1: whole cell protein, lane 2: crude cell extract, lane 3 and 4:

Ni-NTA affinity chromatography and desalting

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9.0

25

8.6

98 8

.687

8.2

11 8

.191

8.1

68

7.6

15

7.5

06 7

.493

7.4

86 7

.474

1.00 0.99

2.04

0.98

1.07

9.0 8.5 8.0 7.5 7.0 PPM

Fig. S4. 1H NMR (dimethylsulfoxide [DMSO], 400 MHz) spectrum of nicotinamide

from 3-cyanopridine catalyzed by recombinant NHase_F1. Nicotinamide was

dissolved in DMSO-D6.

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Fig. S5. ESI-(+)-LC/MS spectrum of nicotinamide from 3-cyanopridine catalyzed by

recombinant NHase_F1. The mass peak with m/z 123.12 corresponds to the [M + H]+

of nicotinamide.

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Fig. S6. Superimposition of NHases from P. putida F1 and P. chlororaphis B23.

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