Backbone Flexibility, Conformational Change, and Catalysis in a Phosphohexomutase from Pseudomonas aeruginosa †,‡ Andrew M. Schramm, § Ritcha Mehra-Chaudhary, § Cristina M. Furdui,* ,| and Lesa J. Beamer* ,§ Departments of Biochemistry and Chemistry, UniVersity of Missouri, Columbia, Missouri 65211, and Department of Internal Medicine, Wake Forest UniVersity Health Sciences, Winston-Salem, North Carolina 27157 ReceiVed March 26, 2008; ReVised Manuscript ReceiVed June 27, 2008 ABSTRACT: The enzyme phosphomannomutase/phosphoglucomutase (PMM/PGM) from the bacterium Pseudomonas aeruginosa is involved in the biosynthesis of several complex carbohydrates, including alginate, lipopolysaccharide, and rhamnolipid. Previous structural studies of this protein have shown that binding of substrates produces a rotation of the C-terminal domain, changing the active site from an open cleft in the apoenzyme into a deep, solvent inaccessible pocket where phosphoryl transfer takes place. We report herein site-directed mutagenesis, kinetic, and structural studies in examining the role of residues in the hinge between domains 3 and 4, as well as residues that participate in enzyme-substrate contacts and help form the multidomain “lid” of the active site. We find that the backbone flexibility of residues in the hinge region (e.g., mutation of proline to glycine/alanine) affects the efficiency of the reaction, decreasing k cat by ∼10-fold and increasing K m by ∼2-fold. Moreover, thermodynamic analyses show that these changes are due primarily to entropic effects, consistent with an increase in the flexibility of the polypeptide backbone leading to a decreased probability of forming a catalytically productive active site. These results for the hinge residues contrast with those for mutants in the active site of the enzyme, which have profound effects on enzyme kinetics (10 2 -10 3 -fold decrease in k cat /K m ) and also show substantial differences in their thermodynamic parameters relative to those of the wild-type (WT) enzyme. These studies support the concept that polypeptide flexibility in protein hinges may evolve to optimize and tune reaction rates. The enzyme PMM/PGM 1 plays a key role in the produc- tion of carbohydrates by Pseudomonas aeruginosa, an opportunistic human pathogen. Three of these molecules are alginate, a secreted exopolysaccharide; lipopolysaccharide, the major component of the outer membrane of the bacte- rium; and rhamnolipid, a surfactant involved in biofilm maintenance (1, 2). Several studies, including animal models of infection, have shown that P. aeruginosa strains lacking PMM/PGM exhibit slower growth rates and decreased virulence and are more easily cleared by the host immune system (3, 4). Hence, PMM/PGM is an attractive target for the development of clinical inhibitors, which could have utility in the treatment of antibiotic resistant infections by this organism. PMM/PGM participates in the early stages of carbohydrate biosynthesis, catalyzing the reversible conversion of 1- to 6-phosphosugars via a bisphosphorylated sugar intermediate (5). It can utilize both glucose- and mannose-based substrates with equal efficiency (6). The reaction entails two phosphoryl transfer reactions: first from a phosphoserine residue on the enzyme to substrate and second from the reaction intermedi- ate (e.g., glucose 1,6-bisphosphate or G16P) back to the enzyme. Previous studies of PMM/PGM have shown that G16P must undergo an ∼180° reorientation between the two phosphoryl transfer steps and that this occurs without dissociation of the intermediate from the enzyme (7, 8). Thus, the PMM/PGM reaction may be considered a simple example of processivity, as defined by multiple rounds of catalysis without release of substrate (9). Several lines of evidence have suggested that conforma- tional change plays a critical role in the reaction of PMM/ PGM. Structural studies of the enzyme, as both apoprotein and in complex with its substrates and products (8, 10), show that an interdomain rotation of the enzyme occurs upon substrate binding, which is necessary to create a high-affinity ligand binding site and position the substrate appropriately for catalysis. In addition, steady-state and pre-steady-state kinetics investigations have substantiated the critical, rate- limiting role of the nonchemical steps of the PMM/PGM reaction (i.e., ligand binding and release and/or conforma- † Supported by a grant from the MU Children’s Miracle Network to L.J.B. The Advanced Light Source is supported by the Director, Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy under Contract DE-AC02-05CH11231. ‡ The structures of the P368G mutant and its complex with substrate have been deposited in the Protein Data Bank (PDB) as entries 3C04 and 3BKQ, respectively. * To whom correspondence should be addressed. L.J.B.: telephone, (573) 882-6072; fax, (573) 884-4812; e-mail, beamerl@missouri.edu. C.M.F.: telephone, (336) 716-2697; fax, (336) 716-1214; e-mail, cfurdui@wfubmc.edu. § University of Missouri. | Wake Forest University Health Sciences. 1 Abbreviations: PMM/PGM, phosphomannomutase/phosphogluco- mutase; DTT, dithiothreitol; MOPS, 3-(N-morpholino)propanesulfonic acid; rmsd, root-mean-square deviation; EDTA, ethylenediaminetet- raacetate; NAD + , nicotinamide adenine dinucleotide (oxidized form); NADH, nicotinamide adenine dinucleotide (reduced form); G1P, glucose 1-phosphate; G16P, glucose 1,6-bisphosphate; WT, wild-type. Biochemistry 2008, 47, 9154–9162 9154 10.1021/bi8005219 CCC: $40.75  2008 American Chemical Society Published on Web 08/09/2008