A Coevolutionary Residue Network at the Site of a Functionally Important Conformational Change in a Phosphohexomutase Enzyme Family

Abstract

Coevolution analyses identify residues that co-vary with each other during evolution, revealing sequence relationships unobservable from traditional multiple sequence alignments. Here we describe a coevolutionary analysis of phosphomannomutase/phosphoglucomutase (PMM/PGM), a widespread and diverse enzyme family involved in carbohydrate biosynthesis. Mutual information and graph theory were utilized to identify a network of highly connected residues with high significance. An examination of the most tightly connected regions of the coevolutionary network reveals that most of the involved residues are localized near an interdomain interface of this enzyme, known to be the site of a functionally important conformational change. The roles of four interface residues found in this network were examined via site-directed mutagenesis and kinetic characterization. For three of these residues, mutation to alanine reduces enzyme specificity to ∼10% or less of wild-type, while the other has ∼45% activity of wild-type enzyme. An additional mutant of an interface residue that is not densely connected in the coevolutionary network was also characterized, and shows no change in activity relative to wild-type enzyme. The results of these studies are interpreted in the context of structural and functional data on PMM/PGM. Together, they demonstrate that a network of coevolving residues links the highly conserved active site with the interdomain conformational change necessary for the multi-step catalytic reaction. This work adds to our understanding of the functional roles of coevolving residue networks, and has implications for the definition of catalytically important residues.

Data on the phosphorylation state of the catalytic serine of enzymes in the α-D-phosphohexomutase superfamily

“…Expression and purification of Pseduomonas aeruginosa PMM/PGM (PaPMM), Bacillus anthracis phosphoglucosamine mutase (BaPNGM), Salmonella typhimurium PGM (StPGM), Francisella tularensis PNGM (FtPNGM), and human phosphoglucosmutase 1 (hPGM1) were performed as described previously [1], [2], [3], [4]. Purified proteins were dialyzed into 50 mM MOPS, pH 7.4, concentrated, and stored at −80 °C until use.…”

“…The conversion of NAD to NADH was monitored by UV–vis spectrophotometry on a CARY 100 spectrophotometer at 25 °C, as previously described [3]. Time courses of enzyme activity in the presence of glucosamine 1-phosphate (GlcN1P) and glucosamine 6-phosphate (GlcN6P) were conducted using 0.14 µM enzyme with 0.5 µM of glucose 1,6-bisphosphate (G16P), and 135 µM of substrate, glucose 1-phosphate (G1P).…”

Chemical shift assignments of domain 4 from the phosphohexomutase from Pseudomonas aeruginosa suggest that freeing perturbs its coevolved domain interface

“…One comparatively simple, processive enzyme utilizing multiple domains is phosphomannomutase/phosphoglucomutase (PMM/PGM), which processes sugars in carbohydrate biosynthesis (Regni et al 2006). Its catalysis (Naught and Tipton 2001, 2005), structure (Regni et al 2002), ligand-binding (Regni et al 2004), evolution (Shackelford et al 2004; Lee et al 2012), and domain movement (Regni et al 2006; Sarma et al 2012; Luebbering et al 2012) have been characterized in a bacterial representative of the α-D-phosphohexomutase superfamily. The enzyme catalyzes reversible phosphoryl transfer between glucose 6-phosphate and glucose 1-phosphate (G1P), or between mannose 6-phosphate and mannose 1-phosphate (M1P) (Ray and Roscelli 1964; Naught and Tipton 2001, 2005).…”

“…Wild-type PMM/PGM limits the flexibility of the hinge between D3 and D4 with a proline in order to optimize activity (Schramm et al 2008). Part of the surfaces of D3 and D4 in contact were recently demonstrated to have coevolved in the PMM/PGM family (Lee et al 2012). …”

“…2b) to be minimal. Among the perturbed positions, L419, V430, and R432 show the highest level of coevolution (Lee et al 2012), implying their functional importance in a network across the interface between D3 and D4 (Lee et al 2012). Also among the perturbed residues of the interface are W417, R432, and F433 which surround the cavity named H2 between D3 and D4 (Chuang et al 2010) (Fig.…”