PDBsum entry 1nsm

Isomerase

PDB id: 1nsm

Contents

Protein chains

339 a.a. *

Ligands

GAL

GLA

Metals

_NA

Waters ×467

* Residue conservation analysis

PDB id:

1nsm

Name:

Isomerase

Title:

Crystal structure of galactose mutarotase from lactococcus lactis mutant d243a complexed with galactose

Structure:

Galactose mutarotase. Chain: a, b. Synonym: aldose 1-epimerase. Engineered: yes. Mutation: yes

Source:

Lactococcus lactis. Organism_taxid: 1358. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Dimer (from PQS)

Resolution:

1.85Å R-factor: 0.179 R-free: 0.236

Authors:

H.M.Holden,J.B.Thoden

Key ref:

J.B.Thoden et al. (2003). The catalytic mechanism of galactose mutarotase. Protein Sci, 12, 1051-1059. PubMed id: 12717027 DOI: 10.1110/ps.0243203

Date:

28-Jan-03 Release date: 11-Feb-03

Protein chains

Q9ZB17  (Q9ZB17_9LACT) -  Aldose 1-epimerase from Lactococcus lactis

Seq: 339 a.a.

Struc: 339 a.a.*

* PDB and UniProt seqs differ at 4 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.5.1.3.3 - aldose 1-epimerase.
• Reaction: alpha-D-glucose = beta-D-glucose

alpha-D-glucose (Bound ligand (Het Group name = GAL) corresponds exactly) = beta-D-glucose

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

Added reference

DOI no: 10.1110/ps.0243203

Protein Sci 12:1051-1059 (2003)

PubMed id: 12717027

The catalytic mechanism of galactose mutarotase.

J.B.Thoden, J.Kim, F.M.Raushel, H.M.Holden.

ABSTRACT

Galactose mutarotase catalyzes the first step in normal galactose metabolism by catalyzing the conversion of beta-D-galactose to alpha-D-galactose. The structure of the enzyme from Lactococcus lactis was recently solved in this laboratory and shown to be topologically similar to domain 5 of beta-galactosidase. From this initial X-ray analysis, four amino acid residues were demonstrated to be intimately involved in sugar binding to the protein: His 96, His 170, Asp 243, and Glu 304. Here we present a combined X-ray crystallographic and kinetic analysis designed to examine the role of these residues in the reaction mechanism of the enzyme. For this investigation, the following site-directed mutant proteins were prepared: H96N, H170N, D243N, D243A, E304Q, and E304A. All of the structures of these proteins, complexed with either glucose or galactose, were solved to a nominal resolution of 1.95 A or better, and their kinetic parameters were measured against D-galactose, D-glucose, L-arabinose, or D-xylose. From these studies, it can be concluded that Glu 304 and His 170 are critical for catalysis and that His 96 and Asp 243 are important for proper substrate positioning within the active site. Specifically, Glu 304 serves as the active site base to initiate the reaction by removing the proton from the C-1 hydroxyl group of the sugar substrate and His 170 functions as the active site acid to protonate the C-5 ring oxygen.

Selected figure(s)

Figure 1.
Figure 1. Active site of galactose mutarotase from L. lactis. A close-up view of the active site within ~5 Å of the galactose ligand is displayed in (A). Ordered water molecules surrounding the sugar ligand were omitted for figure clarity. Both the - and ß-anomers of galactose were observed in the electron density map. A close-up view of the active site within ~5 Å of bound glucose is presented in (B). All figures in this article were prepared with the software package MOLSCRIPT (Kraulis 1991).

Figure 3.
Figure 3. Schematics of hydrogen bonding patterns. Shown in (A) is the observed hydrogen bonding pattern between the wild-type protein and galactose. Both the - and ß-anomers at C-1 are observed in the active site. The hydrogen bonding pattern exhibited between the E304Q mutant protein and glucose is depicted in (B).

The above figures are reprinted by permission from the Protein Society: Protein Sci (2003, 12, 1051-1059) copyright 2003.

Figures were selected by an automated process.