Structure-based Functional Annotation

Abstract

Despite the generation of a large amount of sequence information over the last decade, more than 40% of well characterized enzymatic functions still lack associated protein sequences. Assigning protein sequences to documented biochemical functions is an interesting challenge. We illustrate here that structural genomics may be a reasonable approach in addressing these questions. We present the crystal structure of the Saccharomyces cerevisiae YMR099cp, a protein of unknown function. YMR099cp adopts the same fold as galactose mutarotase and shares the same catalytic machinery necessary for the interconversion of the ␣ and ␤ anomers of galactose. The structure revealed the presence in the active site of a sulfate ion attached by an arginine clamp made by the side chain from two strictly conserved arginine residues. This sulfate is ideally positioned to mimic the phosphate group of hexose 6-phosphate. We have subsequently successfully demonstrated that YMR099cp is a hexose-6-phosphate mutarotase with broad substrate specificity. We solved high resolution structures of some substrate enzyme complexes, further confirming our functional hypothesis. The metabolic role of a hexose-6-phosphate mutarotase is discussed. This work illustrates that structural information has been crucial to assign YMR099cp to the orphan EC activity: hexose-phosphate mutarotase.

Crystal Structures and Enzyme Mechanisms of a Dual Fucose Mutarotase/Ribose Pyranase

“…In comparison, the tyrosine functionality is served by glutamate in GalM and yeast YMR099cp, which uses the same set of catalytic residues as GalM but additionally two arginine residues for binding the phosphate group of sugar substrates. 5,24 Regardless of this difference, the deprotonation and protonation of C1-OH are proposed to be achieved by a single The numbers in parentheses are statistics from the highestresolution shell. a Thr4 in the seven subunits are the only residues in the generously or disallowed regions, but their electron densities are well defined.…”

“…In comparison, YMR099cp was crystallized in complex with tagatose 6-phosphate (the furan form of galactose 6-phosphate), which was present in a trace amount together with galactose 6-phosphate. 5 Since tagatose 6-phosphate does not undergo configurational change at C1, the enzyme's unusual selectivity for this sugar is elusive.…”

“…3,4 Through structureinspired biochemical analyses, a hypothetical yeast protein, YMR099cp, was characterized as D-hexose 6-phosphate mutarotase. 5 Recently, RhaU of Rhizobium leguminosarum, which exhibits weak sequence homology with YiiL, was characterized as an L-rhamnose mutarotase. 6 In addition, RbsD, which exhibits 25% sequence identity with FucU, was shown to catalyze the interconversion between the five-and six-membered ring forms of D-ribose.…”

The Crystal Structure of Trypanosoma cruzi Glucokinase Reveals Features Determining Oligomerization and Anomer Specificity of Hexose-phosphorylating Enzymes

“…Well known examples of mutarotases are aldose 1-epimerase, which shows a fourfold higher catalytic constant for the reaction with D-galactose over D-glucose anomers, 29 and hexose-6-phosphate mutarotase, which catalyzes the mutarotation between D-glucose 6-phosphate anomers. 30 On the basis of sequence identity (50%) with yeast hexokinase, and a complete conservation of glucoseinteracting residues, a preference for the α anomer is expected for T. cruzi hexokinase. 28 In contrast to the hexokinases, our kinetic and structural data now indicate a moderate preference of the TcGlcK for the β anomer of glucose.…”

Through the Looking Glass: Chiral Recognition of Substrates and Products at the Active Sites of Racemases and Epimerases

“…This effects the epimerizationa t the C1 carbon atom to furnish the appropriate substrate for other enzymes acting on reducing sugars that are specific for a single anomer.S everal structures of mutarotases with bound substrateh ave been reported. Often the bound substrate is a single anomer; [244][245][246][247][248][249] however, in somec ases, the observed electron density is consistent with the presence of both the aand b-anomers of the substrate presenti nt he crystalline enzyme.T he latter cases are discussed below.…”