Disulfide-linked dimers of human adrenaline synthesizing enzyme PNMT are catalytically active.
The crystal structure of human phenylethanolamine N-methyltransferase (hPNMT) reveals a disulfide-linked dimer, despite the presence of reducing agent in the crystallisation conditions. By removing the reducing agent, hPNMT crystals grow more rapidly and at lower protein concentrations. However, it was unclear whether the disulfide bonds are only present in the crystal form or whether these affect enzyme activity. The solution oligomeric state of hPNMT was investigated using biochemical techniques and activity assays. We found that in the absence of reducing agent, hPNMT forms dimers in solution. Furthermore, the solution dimer of hPNMT incorporates disulfide bonds, since this form is sensitive to reducing agent. The C48A and C139A mutants of hPNMT, which are incapable of forming the disulfide bond observed in the crystal structure, have a decreased propensity to form dimer in solution. Those dimers that do form are also sensitive to reducing agent. Further, the C48A/C139A double mutant shows only monomeric behaviour. Both dimeric and monomeric hPNMT, as well as mutants have wildtype enzyme activity. These results show that a variety of disulfides, including those observed in the crystal structure, can form in solution. In addition, disulfide-linked dimers are as active as the monomeric enzyme indicating that the crystal structure of the protein is a valid target for inhibitor design.
Gee
Christine L
CL
Institute for Molecular Bioscience and ARC Special Research Centre for Functional and Applied Genomics, University of Queensland, Brisbane Qld, 4072 Australia.
Nourse
Amanda
A
Hsin
A-Yen
AY
Wu
Qian
Q
Tyndall
Joel D
JD
Grunewald
Gary L
GL
McLeish
Michael J
MJ
Martin
Jennifer L
JL
D001665:Binding Sites; D002384:Catalysis; D003460:Crystallization; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000235:genetics; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry*; D004229:Dithiothreitol / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D010625:Phenylethanolamine N-Methyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D019163:Reducing Agents / Q000737:chemistry / Q000378:metabolism; D012996:Solutions; D014461:Ultracentrifugation / Q000379:methods
D004220:Disulfides; D019163:Reducing Agents; D012996:Solutions; D010625:Phenylethanolamine N-Methyltransferase; D003545:Cysteine; D004229:Dithiothreitol