General Information

Top of Page

Combining Crystallography and Visible Spectroscopy to Understand Enzymes

stachydrine demethylase from Sinorhizobium meliloti 1021

The research team focused on the stachydrine demethylase from Sinorhizobium meliloti 1021. The applications of electronic absorption and resonance Raman spectroscopies, in conjunction with x-ray diffraction, all derive from the same ~25 µm2 region of the crystal (intersection of the cross hairs). The x-ray beam alters the visible spectrum of the crystal with each exposure required to determine the crystal structure of the trimeric enzyme and the Fe-S cluster within each subunit.

Structure and function are intimately linked, but do not necessarily predict the other. For example, x-ray crystallography provides 3D atomic structural information about biological macromolecules but does not define important details about metal ions.

However, the oxidation state of metal ions at an enzyme’s active site has a critical effect on enzyme behavior. Thus, an enzyme’s catalytic function derives from the electronic structure of those atoms influencing or directly participating in the reaction, information not revealed by the scattering methods used in x-ray crystallography.

A new technology has been developed that simultaneously carries out crystallography and UV-visible and Raman spectroscopy to determine the atomic structure of the entire protein, and electronic and vibrational structures of the metal ions or cofactors within. The combined instrumentation has been used to study the process of demethylation of an organic substrate molecule by an enzyme whose active site includes an iron-sulfur cluster.

The researchers used spectroscopy to follow the change in the oxidation state of the cluster during the crystallography data collection, and to formulate a mechanism for the overall enzyme reaction mechanism. The results provide insight into a new demethylation reaction, an important class of phenomena that control cellular behavior.

The technology was developed by scientists at the Protein Crystallography Research Resource at the National Synchrotron Light Source at Brookhaven National Laboratory. The new study was led by Allen M. Orville of Brookhaven and Pinghua Liu and Karen N. Allen of Boston University, and is published in the Journal of the American Chemical Society.

Funding: This work was supported by the Office of Biological and Environmental Research, US Department of Energy (FWP BO-70), and the National Institutes of Health, National Institute for General Medical Sciences, Biomedical Technology Branch (8P41GM103473). Data were measured at beamline X26C of the National Synchrotron Light Source (NSLS) at the Brookhaven National Laboratory. Use of the NSLS was supported by the US Department of Energy Office of Basic Energy Sciences, under Contract DE-AC02-98CH10886.

Contact: Allen M. Orville, amorv@bnl.gov or 631-344-4739

https://public.ornl.gov/site/bernews/default.cfm

2012-3175  INT/EXT  |  Media & Communications Office

Subscribe

You can recieve a monthy reminder email when a new issue of eNews is available. If you are not already receiving this email, then subscribe below.

Your email address

Your name (optional)

Subscribe

You will receive a email asking you to confirm that you want to subscribe to this list. If you don't see this email in your inbox, then check your spam or junkmail box.

Once subscribed to this service, you will receive an monthly email announcing the availability of that month's edition of eNews. Your subscription will be used solely to email you Photon Sciences eNews. Your email address will not be placed on any other mailing lists and will not be used for commercial purposes.