High Resolution X-ray Structure of Galactose Mutarotase from Lactococcus lactis*

Galactose mutarotase plays a key role in normal galactose metabolism by catalyzing the interconversion of β-d-galactose and α-d-galactose. Here we describe the three-dimensional architecture of galactose mutarotase from Lactococcus lactis determined to 1.9-Å resolution. Each subunit of the dimeric enzyme displays a distinctive β-sandwich motif. This tertiary structural element was first identified in β-galactosidase and subsequently observed in copper amine oxidase, hyaluronate lyase, chondroitinase, and maltose phosphorylase. Two cis-peptides are found in each subunit, namely Pro⁶⁷ and Lys¹³⁶. The active site is positioned in a rather open cleft, and the electron density corresponding to the bound galactose unequivocally demonstrates that both anomers of the substrate are present in the crystalline enzyme. Those residues responsible for anchoring the sugar to the protein include Arg⁷¹, His⁹⁶, His¹⁷⁰, Asp²⁴³, and Glu³⁰⁴. Both His⁹⁶ and His¹⁷⁰ are strictly conserved among mutarotase amino acid sequences determined thus far. The imidazole nitrogens of these residues are located within hydrogen bonding distance to the C-5 oxygen of galactose. Strikingly, the carboxylate group of Glu³⁰⁴ is situated at ∼2.7 Å from the 1′-hydroxyl group of galactose, thereby suggesting its possible role as a general acid/base group.