Human methylmalonyl Coenzyme A mutase

Target ID:

MUTA

Aliases:

MCMMUT

Deposition Date:

Friday 30th November 2007

Families:

Miscellaneous

Related Diseases:

MetabolismNeurobiology

Structure type:

apo

Download Coordinates:

3BIC

Authors:

Ugochukwu, E. Kochan, G. Pantic, N. Parizotto, E. Pike, A. C. W. Gileadi, O. von Delft, F. Arrowsmith, C. H. Weigelt, J. Edwards, A. Oppermann, U.

Site:

Oxford

3BIC

tabs

Structure Details

The degradation of the amino acids methionine, isoleucine and valine, as well as the β-oxidation of odd-chain fatty acids, results in the formation of propionyl-CoA. This intermediate needs to be transformed in a 3-step process to succinyl-CoA for further breakdown in the TCA cycle. The first step in this sequence is catalyzed by propionyl-CoA carboxylase, resulting in D-methylmalonyl CoA, which is further converted by a racemase to L-methylmalonyl CoA, which is the substrate for methylmalonyl CoA mutase. This enzyme converts in a Vitamin B12 dependent, free radical mediated reaction its substrate to succinyl-CoA.

Mutations in the human methylmalonyl-CoA mutase gene lead to methylmalonic acidemia, a disorder of organic acid metabolism in which precursors and abnormal metabolites of L-methylmalonyl-CoA can cause widespread aberrations in metabolic homeostasis.

MUT deficiency may affect as many as 1/29,000 newborns and the clinical phenotypes observed range from fulminant neonatal acidosis and death, to benign, asymptomatic methylmalonic aciduria. Common clinical features include lethargy, vomiting, failure to thrive, hypotonia, neurological deficits and early death.

Materials and Methods

MUT

PDB:3BIC

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi|4557767
Entry Clone Source:MGC
SGC Clone Accession:IMAGE:3908548
Tag:C-terminal hexahistidine tag and Flag tag, TEV cleavable: aenlyfq*shhhhhhdykddddk
Host:BL21(DE3)-R3-pRARE2

Construct

Prelude:
Sequence:MSPHYLRQVKESSGSRLIQQRLLHQQQPLHPEWAALAKKQLKGKNPEDLIWHTPEGISIKPLYSKRDTMDLPEELPGVKPFTRGPYPTMYTFRPWTIRQYAGFSTVEESNKFYKDNIKAGQQGLSVAFDLATHRGYDSDNPRVRGDVGMAGVAIDTVEDTKILFDGIPLEKMSVSMTMNGAVIPVLANFIVTGEEQGVPKEKLTGTIQNDILKEFMVRNTYIFPPEPSMKIIADIFEYTAKHMPKFNSISISGYHMQEAGADAILELAYTLADGLEYSRTGLQAGLTIDEFAPRLSFFWGIGMNFYMEIAKMRAGRRLWAHLIEKMFQPKNSKSLLLRAHCQTSGWSLTEQDPYNNIVRTAIEAMAAVFGGTQSLHTNSFDEALGLPTVKSARIARNTQIIIQEESGIPKVADPWGGSYMMECLTNDVYDAALKLINEIEEMGGMAKAVAEGIPKLRIEECAARRQARIDSGSEVIVGVNKYQLEKEDTVEVLAIDNTSVRNRQIEKLKKIKSSRDQALAERCLAALTECAASGDGNILALAVDASRARCTVGEITDALKKVFGEHKANDRMVSGAYRQEFGESKEITSAIKRVHKFMEREGRRPRLLVAKMGQDGHDRGAKVIATGFADLGFDVDIGPLFQTPREVAQQAVDADVHAVGVSTLAAGHKTLVPELIKELNSLGRPDILVMCGGVIPPQDYEFLFEVGVSNVFGPGTRIPKAAVQVLDDIEKCLEKKQQSVaenlyfq*shhhhhhdykddddk
Vector:pNIC-CTHF

Growth

Medium:
Antibiotics:
Procedure:10µl of BL21(DE3)-R3 glycerol stock were inoculated into 5ml of Terrific broth medium supplemented with kanamycin (50 µg/ml) and chloramphenicol (34µg/ml) and grown overnight at 37°C, 200rpm. In the morning 1L of TB supplemented with the same antibiotics was inoculated with 10ml of the overnight culture and incubated at 37°Cwith intensive shaking (160rpm). After the OD600 reached 1.5, the temperature was changed to 18°C and IPTG was added to the final concentration of ~0.1mM. The culture was incubated at 18°C with shaking (160rpm) for additional 18h. The following morning the 3 l culture was harvested and centrifuged for 10min at 4000rpm. Supernatant was discarded and the cell pellets were resuspended in 75ml of a lysis buffer and frozen at -80°C.

Purification

Procedure
Step 1: Ni-affinity, Ni-Sepharose - (GE Healthcare) Acta-Express; Step 2: Superdex 200 Column, HiPrep 16/60 (Amersham); Step 3: Ion exchange -5ml HiTrap Q SepharoseThe cell extract was loaded on the AKTA Express system The extinction at 280nm was monitored and fractions were collected and analyzed by SDS-PAGE. MUTA containing fractions were diluted with IEX buffer A to a concentration of NaCl of 50 mM. The protein was loaded onto a HiTrap Q Sepharose column, and eluted with a NaCl gradient (50-300mM). Fractions containing protein were analysed by SDS-PAGE.

Extraction

Procedure
The thawed cells were broken by 5 passes at 16.000 psi through a high pressure homogeniser, followed by centrifugation for 45 min at 20.000rpm at 4°C.
Concentration:Using Amicon Ultra-15 concentrators with 30kDa cutoff, the sample was concentrated to 11mg/ml. Concentrations were determined from the absorbance at 280 nm using a NanoDrop spectrophotometer.
Ligand
MassSpec:The calculated mass of the construct was 84724Da, and the observed mass (ESI-MS) was 84510Da, compatible with an N-terminal methionine and serine deletion.
Crystallization:Crystals were grown by vapor diffusion at 20°C in 150nl sitting drops. Vitamin B12 was added to a final concentration of 5mM prior to crystallisation. The drops were prepared by mixing 50nl of protein solution and 100nl of precipitant consisting of 1.60M Na/KPO4; 0.1M HEPES pH 7.5. Crystals were flash-cooled in liquid nitrogen with 25% glycerol as cryoprotectant.
NMR Spectroscopy:
Data Collection:Resolution: 2.65Å; X-ray source: Swiss Light source (SLS), beamline X-10.
Data Processing: