X-Ray Crystallography of 2-methyl-3-hydroxypyridine-5-carboxylic acid Oxygenase

Speaker: Stuart T. Streeter

Abstract: Understanding an enzyme mechanism operate requires detailed information that defines at the atomic level the enzyme’s tertiary structure. Because of the minute scale and delicacy of protein structures involved, individual atoms being in the one angstrom (Å) range and enzymes extremely susceptible to changes in environment, common techniques for gathering data cannot be employed. At the most fundamental level, the wavelengths of so-called “visible light” and the ultra-violet spectrums cannot be resolved to provide data on atomic structures. While nuclear magnetic resonance imaging (NMR imaging) has solved some protein structures, this technique is limited to proteins with a molecular mass below approximately 35 kDa. Many proteins are far above this limit and many more are functional only in multimers that also exceed this limit. Structure data is of minimal use in understanding an enzyme’s function if the structure solved is not the active configuration.

X-ray crystallography is able bypass these limitations. First, in using x-rays, which have a wavelength in the 1 Å range, data can be resolved to provide information at the atomic level. Secondly, protein crystals provide the repeated and orderly arrangement of molecules required to collect sufficient data to begin structure determination. Additionally, crystallized proteins generally retain their function and can be quickly tested to assure this is the case simply by exposing the crystallized protein to its substrate and searching for the product(s).

I am currently working to solve the structures of several different proteins. 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase (MHPCO) catalyzes the breakdown of MHPC aka Vitamin B6 aka pyridoxine to AAMS, a readily utilized metabolite and is found in several of the Pseudomonas genus of bacteria playing a central role in uric acid metabolism. MHPCO is my first project and the one that I have made the most progress in thus far. I will be speaking today about the stages I have reached in the structure determination of MHPCO, the methods entailed, and significant results.