The ␣-D-phosphohexomutase superfamily is composed of four related enzymes that catalyze a reversible, intramolecular phosphoryl transfer on their sugar substrates. The enzymes in this superfamily play important and diverse roles in carbohydrate metabolism in organisms from bacteria to humans. Recent structural and mechanistic studies of one member of this superfamily, phosphomannomutase/phosphoglucomutase (PMM/ PGM) from Pseudomonas aeruginosa, have provided new insights into enzyme mechanism and substrate recognition. Here we use sequence-sequence and sequence-structure comparisons via evolutionary trace analysis to examine 71 members of the ␣-D-phosphohexomutase superfamily. These analyses show that key residues in the active site, including many of those involved in substrate contacts in the P. aeruginosa PMM/PGM complexes, are conserved throughout the enzyme family. Several important regions show class-specific differences in sequence that appear to be correlated with differences in substrate specificity exhibited by subgroups of the family. In addition, we describe the translocation of a 20-residue segment containing the catalytic phosphoserine of phosphoacetylglucosamine mutase, which uniquely identifies members of this subgroup.Keywords: phosphohexomutase; evolutionary trace; enzyme superfamily; carbohydrate metabolismThe ␣-D-phosphohexomutase enzyme superfamily is widespread and diverse. Two related and well-characterized proteins make up the majority of the family: the highly specific PGM, which only uses glucose as a substrate, and the less specific PMM/PGM, which can use either glucose or mannose. Two other enzymes, PNGM and PAGM, are also members of the superfamily, although to date these two proteins have been less extensively characterized. The ␣-Dphosphohexomutases play varied roles in carbohydrate metabolism and other biosynthetic pathways. PGM is best known for its role in providing substrates that enter the glycolytic pathway. The PMM/PGM proteins are primarily bacterial and participate in the biosynthesis of a variety of carbohydrates, such as lipopolysaccharide and alginate (Shankar et al. 1995;Rocchetta et al. 1999). PNGM and PAGM are involved in the biosynthesis of UDP-N-acetylglucosamine, which is an essential common precursor for bacterial cell wall components and is also required for the posttranslational N-acetylglucosamine modification of eukaryotic proteins (Jolly et al. 1999;Mio et al. 2000).Despite differences in substrate specificity, the enzymes in this superfamily that have been characterized to date appear to use the same mechanism. They catalyze the reversible conversion of 1-phospho to 6-phosphosugars via a bisphosphorylated sugar intermediate. Active enzyme is phosphorylated at a conserved serine residue and binds one Mg 2+ ion. The reaction mechanism entails two phosphoryl transfer reactions: first, from the enzyme to substrate, and second, from the reaction intermediate back to the enzyme. The initial phosphoryl transfer is from phosphoserine to bound substrate, cre...

PDB References: PMM/PGM-R1P complex, 2h4l, r2h5lsf; PMM/PGM-X1P complex, 2h5a, r2h5asf.Two complexes of the enzyme phosphomannomutase/phosphoglucomutase (PMM/PGM) from Pseudomonas aeruginosa with a slow substrate and with an inhibitor have been characterized by X-ray crystallography. Both ligands induce an interdomain rearrangement in the enzyme that creates a highly buried active site. Comparisons with enzyme-substrate complexes show that the inhibitor xylose 1-phosphate utilizes many of the previously observed enzyme-ligand interactions. In contrast, analysis of the ribose 1-phosphate complex reveals a combination of new and conserved enzyme-ligand interactions for binding. The ability of PMM/PGM to accommodate these two pentose phosphosugars in its active site may be relevant for future efforts towards inhibitor design.

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