Human Carbonyl Reductase

Target ID:

CBR3A

Aliases:

hCBR3

Deposition Date:

Thursday 20th July 2006

Families:

Oxidoreductases

Related Diseases:

NeurobiologyCancerMetabolismDrug metabolism and toxicology

Structure type:

apo

Download Coordinates:

2HRB

Authors:

E.S.PILKA,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Oxford

ICM Datapack:

ICM Datapack

2HRB

tabs

Structure Details

Human carbonyl reductase (CBR) activity accounts for a significant fraction of the metabolism of endogenous and xenobiotic carbonyl compounds such as prostaglandins, steroids, and various pharmacological agents. CBRs are members of the family of short-chain dehydrogenases/reductases. In humans, there are two monomeric carbonyl reductases, carbonyl reductase 1 (CBR1) and carbonyl reductase 3 (CBR3), which are encoded by different genes located 62 kilobases apart on chromosome 21 (CBR1 and CBR3). CBR1 and CBR3 share 72% identity and 79% similarity at the amino acid level.

Despite the prominent role of CBR1 mediated drug metabolism, there have been few reports on the catalytic properties of CBR3 since the identification of the gene in 1998, in particular no endogenous substrate has been identified up to date. Several studies have described a wide range of variability in the metabolism of drugs that are CBR substrates, which may contribute to the largely unpredictable pharmacokinetics and pharmacodynamics of anthracyclines in adult and pediatric cancer patients. It is possible that genetic polymorphisms in CBR1 and CBR3 are the key for the variability in the disposition of CBR drug substrates.

Materials and Methods

CBR3: Human Carbonyl Reductase 3

PDB:2HRB

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:CBR3A-s001
Entry Clone Source:MGC
SGC Clone Accession:
Tag:
Host:E.coli strain BL21(DE3)-R3

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfq*sMSSCSRVALVTGANRGIGLAIARELCRQFSGDVVLTARDVARGQAAVQQLQAEGLSPRFHQLDIDDLQSIRALRDFLRKEYGGLNVLVNNAAVAFKSDDPMPFDIKAEMTLKTNFFATRNMCNELLPIMKPHGRVVNISSLQCLRAFENCSEDLQERFHSETLTEGDLVDLMKKFVEDTKNEVHEREGWPNSPYGVSKLGVTVLSRILARRLDEKRKADRILVNACCPGPVKTDMDGKDSIRTVEEGAETPVYLALLPPDATEPQGQLVHDKVVQNW
Vector:pNIC28-Bsa4

Growth

Medium:
Antibiotics:
Procedure:10 µl BL21(DE3)-R3 glycerol stock were inoculated into 5ml of TB with 50µg/ml of kanamycin and 34µg/ml chloramphenicol and grown overnight at 37°C, 200rpm. 10ml of overnight culture were added to 1L of TB with 50ug/ml kanamycin and incubated at 37°C, 160rpm. After the OD600 reached 1.0, the temperature was dropped to 18°C and 500 µl of 1M IPTG was added to the final concentration of ~0.5mM. The culture was then incubated with shaking overnight at 18°C, 160rpm. The following morning the 4L culture was harvested and centrifuged for 10min at 4000rpm. Supernatant was discarded and cell pellets were resuspended in 80ml of a lysis buffer and frozen at -80°C.

Purification

Procedure
Column 1 : Ni-affinity, His-Trap, 1ml (Amersham)
Column 2 : SuperDex 200 16/60 HiLoad (GE/Amersham)
Buffers: Start buffer: 50mM HEPES pH 7.5, 500mM NaCl, 5mM Imidazole, 5% glycerol, 1mM PMSF, 0.5mM TCEP
Washing buffer: 50mM HEPES pH 7.5, 500mM NaCl, 5mM Imidazole, 5% glycerol, 1mM PMSF, 0.5mM TCEP
Elution buffer: 50mM HEPES pH 7.5, 500mM NaCl, 5% glycerol, 30mM Imidazole, 0.5mM TCEP
GF buffer: 50mM HEPES pH 7.5, 500mM NaCl, 5% glycerol, 0.5mM TCEP
Procedure (Columns 1 and 2): The cell extract was loaded on the AKTA Express system The extinction at 280nm was monitored and fractions were collected and analyzed by SDS-PAGE. Positive fractions were pooled for TEV cleavage.

TEV cleavage : The His-tag was cleaved with 1 mg TEV per 40 mg target protein at 4°C overnight. The protein was purified on IMAC Sepharose using buffers as above.

Column 3 : Desalting column
Column 4 : Ion exchange QHP
Buffers: Buffer A: 50mM HEPES pH 7.5, 50mM NaCl, 0.5mM TCEP
Buffer B: 50mM HEPES pH 7.5, 2M NaCl, 0.5mM TCEP

Procedure (Columns 3 and 4): Desalted protein was purified in a 25% A/B gradient ran over 20 column volumes on QHP. A single resulting peak was characterised by the mass spec

Concentration: Concentration and buffer exchange: Using Amicon Ultra-15 concentrators with 10kDa cutoff, the sample was buffer-exchanged into GF buffer and concentrated to 10mg/ml. Concentrations were determined from the absorbance @ 280nm using NanoDrop.

Extraction

Procedure: The thawed cells were broken by 5 passes at 16,000 psi through a high pressure homogeniser followed by centrifugation for 45min at 15,000rpm.
Concentration:
Ligand:
MassSpec: Calculated mass of the construct was 30937. The exact mass was confirmed by the mass spec
Crystallization:
NMR Spectroscopy :
Data Collection: Resolution: 1.9Å; X-ray source: Rotating anode, Rigaku FR-E superbright .
Data Processing: