Structural Studies of Two Enzymes of Pantothenate Biosynthesis in Escherichia Coli

Change log
Schmitzberger, Florian Johannes 

Pantothenate (vitamin B5), which is the invariable metabolic precursor to coenzyme A, is synthesized from L-aspartate and alpha-ketoisovalerate in a converging four-step process in bacteria. Here, structural studies of two enzymes of pantothenate biosynthesis in Escherichia coli, L-aspartate-alpha-decarboxylase and ketopantoate hydroxymethyltransferase, are described.

Ketopantoate hydroxymethyltransferase catalyzes the transfer of a hydroxymethyl group on to alpha-ketoisovalerate, assisted by the cofactor 5,10-methylene-5,6,7,8-tetrahydrofolate. In order to determine the mode of cofactor binding to the protein, ketopantoate hydroxymethyltransferase was crystallized in the presence of two 5,10-methylene-5,6,7,8-tetrahydrofolate analogues and alpha-ketoisovalerate. X-ray diffraction patterns, collected on the in-house X-ray diffraction data collection facility, extended to 4.0 Angstroem. Unit cell dimensions derived from these diffraction patterns indicate an asymmetric unit with one decameric enzyme. A detailed comparative structural analysis of the fold of ketopantoate hydroxymethyltransferase was carried out. Based on this investigation it was possible to assign the enzyme to the phosphoenolpyruvate/pyruvate enzyme superfamily. Furthermore, similarities in the mode of ligand binding to the catalytic magnesium, as well as differences in the mechanisms between the enzymes within this superfamily could be delineated. In common with a small, but widely distributed, group of mechanistically-related enzymes, L-aspartate-alpha-decarboxylase is translated as an inactive pro-enzyme, which self-processes at a specific site. In this process of intra-molecular protein maturation a covalently bound pyruvoyl cofactor is formed. A fast purification system for eight L-aspartate-alpha-decarboxylase mutants was established that allows production of large amounts of enzyme. In order to gain insights into the molecular mechanism of self-processing, crystallographic studies were carried out. Several of the purified mutants have been crystallized. X-ray diffraction data from glycine 24 to serine and serine 25 to threonine mutants were collected, to a maximum resolution of 1.26 Angstroem. The respective crystal structures were solved by molecular replacement. Along with the structures of an unprocessed, native precursor form of L-aspartate-alpha-decarboxylase and a serine 25 to alanine mutation, the structure models were refined and evaluated, and the models deposited in the Protein Data Bank. Analysis of these four structures together with four other L-aspartate-alpha-decarboxylase mutant structures revealed specific conformational constraints on the self-processing mechanism. Threonine 57 and a water molecule could be identified as catalytic elements, most likely essential for acid-base catalysis, and stabilization of the oxyoxazolidine intermediate in the self-processing reaction. A molecular mechanism for self-processing in L-aspartate-alpha-decarboxylase, largely based on the threonine 57 and a water molecule, is proposed. The differences in the structures of the cleavage site of the serine 25 to alanine and serine 25 to threonine mutants, relative to the structure of the unprocessed native precursor, suggest that molecular models of the cleavage site and mechanisms, based solely on serine to alanine and serine to threonine mutants, may lead to erroneous interpretations of the mechanism. On comparison with other self-processing systems, particularly, glycosylasparaginase, remarkable parallels in the structural features of the environment of the cleavage site were identified.

Blundell, Thomas Leon
Pantothenate (vitamin B5) biosynthesis, Enzymology, Structural biology, Protein crystallography, Protein self-processing, L-aspartate-alpha-decarboxylase, Ketopantoate hydroxymethyltransferase
Doctor of Philosophy (PhD)
Awarding Institution
University of Cambridge
Cambridge European Trust, University of Vienna, Sidney Sussex College