Photon Factory Activity Report 2019 #37 (2020)

BL-5A/2019G015

Crystal structure of galactose 1-phosphate uridylyltransferase
Tatsuya Ohshida 1, *, Junji Hayashi 2, Kazunari Yoneda 3, Toshihisa Ohshima 4, Haruhiko Sakuraba 1
1
Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Miki-cho,Kagawa, Japan
2
Faculty of Bioscience and Bioindustry, Tokushima University, Tokushima, Japan3
Department of Bioscience, School of Agriculture, Tokai University, Kumamoto, Japan
4
Department of Biomedical Engineering, Faculty of Engineering, Osaka Institute of Technology,Osaka, Japan
1 Introduction
In the Leloir pathway of galactose metabolism,galactose is first phosphorylated to galactose 1-phosphate(Gal1P) by galactokinase. The second enzyme, galactose
1-phosphate uridylyltransferase (GalT; EC 2.7.7.12),
catalyzes the transfer of the uridylyl group from UDPglucose (UDP-Glu) to Gal1P to produce UDP-galactose(UDP-Gal) and glucose 1-phosphate (Glu1P). The thirdenzyme in the pathway, UDP-galactose 4-epimerase(GalE; EC 5.1.3.2), regenerates UDP-Glu from UDP-Gal
to maintain the production of Glu1P, which enters theglycolytic pathway. These three enzymes, comprising theLeloir pathway, are widely distributed among bothBacteria and Eukarya, and structural and functional
analyses have been reported. With regard to Archaea, thethird domain of life, galactokinases from thehyperthermophilic archaea Pyrococcus furiosus and P.
horikoshii have been characterized, and the molecularbasis of their substrate recognition has been revealed.
However, information about the other two enzymes
involved in the Leloir pathway in Archaea remains limited.
Our database search revealed the presence ofhomologues of all three enzymes among phylogeneticallydistant archaeal species, including Pyrobaculum andPyrococcus. This suggests that the Leloir pathway isfunctional in hyperthermophilic archaea, and thatstructural and functional analysis of its constituents could
shed light on their features and diversity in these organisms.
We previously identified a gene (Pcal_0885) encoding a
GalE homologue within the genomic sequence of thehyperthermophilic archaeon Pyrobaculum calidifontis andconfirmed the gene product to be an extremely
thermostable GalE. We also solved the crystal structure ofthis enzyme and evaluated the structural features
responsible for its high thermostability. We next identified
a gene (PAE1184) encoding a GalT homologue within thegenome of Pyrobaculum aerophilum (a species closely
related to P. calidifontis) and performed the crystallizationand preliminary X-ray analysis of the gene product. At thattime, we confirmed that the gene product exhibits a highlevel of GalT activity, but were unable to obtain useful data
for structure determination. In the present study, we solved
the crystal structure of P. aerophilum GalT and found thatits active site components are completely different thanthose observed in the two previously reported GalTstructures. Here we present the first crystal structure of anovel type of GalT found in a hyperthermophile in thearchaeal domain and report the formation of a uniqueactive site in this enzyme.
2 Experiment
Data were collected under cryo conditions at theBeamline BL-5A at Photon Factory in Japan. The program
Phaser-MR in the PHENIX was used for molecularreplacement phase determination. The crystal structure ofGalT from Escherichia coli (PDB ID: 1gup, amino acidsequence identity: 30%) was served as the search model.
Model building was performed using the program Coot,and refinement was carried out using REFMAC5.
3 Results and Discussion
Two different crystal structures of P. aerophilum GalT
were determined: the substrate-free enzyme at 2.33 Å andthe UDP-bound H140F mutant enzyme at 1.78 Å. The
main-chain coordinates of the P. aerophilum GalT
monomer were similar to those in the structures of the E.
coli and human GalTs, as was the dimeric arrangement.
However, there was a striking topological difference
between P. aerophilum GalT and the other two enzymes.
In the E. coli and human enzymes, the N-terminal chain
extends from one subunit into the other and forms part ofthe substrate-binding pocket in the neighboring subunit.
By contrast, the N-terminal chain in P. aerophilum GalT
extends to the substrate-binding site in the same subunit(Fig. 1). Amino acid sequence alignment showed that ashorter surface loop in the N-terminal region contributes tothe unique topology of P. aerophilum GalT.
To determine the structure of the E. coli GalT/UDP-Glucomplex, a catalytically inactive mutant, H166G, wasprepared and cocrystallized with UDP-Glu. His166 in E.
coli GalT is conserved as His140 in P. aerophilum GalT.
Therefore, to solve the structure of P. aerophilum GalT
with bound substrate, we prepared H140G, H140N, andH140F mutants of the enzyme and confirmed these threemutants were all inactive. We next endeavored tococrystallize the three mutants with UDP-Glu, collect thediffraction data, and determine their structures. In theelectron-density map for the structure solved with theH140F/UDP-Glu crystal, the electron density for the UDPPhoton Factory Activity Report 2019 #37 (2020)

Fig. 1: Ribbon plots of the main-chain coordinates (green)of, A, E. coli GalT (PDB code: 1guq); B, human GalT(PDB code: 5in3); and C, P. aerophilum GalT (PDB code:6k5z) monomers.
molecule was observed within the active sites of bothsubunits. Although the electron density for the αphosphoryl group was discontinuous, the significant
electron densities at the uridyl ribose, β-phosphoryl group,and uridine moiety enable us to deduce a plausible
structure for UDP (Fig. 2). The structure was determinedat a resolution of 1.78 Å. No density corresponding to theglucose moiety was detected, though the enzyme wascocrystallized with UDP-Glu. Within our model, theuridine moiety of UDP interacts with a backbone N atomof Arg51 (Asp78 in E. coli GalT). To accommodate theuridine moiety, the side chain of Arg51 in UDP-bound
H140F rotates clockwise by about 80° around the Cβ atomof Arg51 relative to the substrate-free P. aerophilum GalTstructure. The 2’- and 3’-hydroxyl groups of the uridyl
ribose form hydrogen bonds with the side chain of Asn50(Asn77 in E. coli GalT). The β-phosphoryl oxygen
interacts with the side chains of Gln142 (Gln168 in E. coliGalT) and Tyr144. Nearly all of these interactions areconserved in the E. coli GalT/UDP-Glu complex, except
for the interaction between the side chain of Tyr144 andthe substrate, as this residue is replaced by Trp170 in E.
coli GalT. In addition, UDP moieties show similar
orientation in both the structures except for the situation ofα- and β-phosphoryl groups. The N-terminal loop,
including Arg19 and Arg22, is disordered in both subunitsof the H140F/UDP structure. It is noteworthy that otherconserved active site residues in H140F/ UDP are situatedat positions similar to those in the substrate-free P.
aerophilum GalT structure (rmsds [average] of 0.33 for theCα atoms of Asn50, Asn127, Gly133, Ser135, Gln142,
Lys280*, and Glu286*)(an asterisk indicates a residue inthe neighboring subunit). This suggests that the binding of
the glucose moiety of the substrate, but not the UDP
moiety, gives rise to a large structural change around theactive site, which in turn provides an appropriate
environment for the enzyme reaction.

Fig. 2: Comparison of the UDP-binding site structuresin substrate-free P. aerophilum GalT (green) and the UDPbound H140F mutant (cyan). The UDP molecule in H140F
is drawn in yellow.
Acknowledgement
We are grateful to the staff of the Photon Factory for
their assistance with data collection, which was approvedby the Photon Factory Program Advisory Committee.
Reference[1] T. Ohshida et al., Proteins 88(5), 669-678 (2020).
Research Achievements
1. T. Ohshida et al., The 2019 Annual Meeting of The
Japan Society for Bioscience, Biotechnology, andAgrochemistry West Japan and Chugoku Shikoku
Branch, E-p06 (2019)
2. T. Ohshida et al., The 2020 Annual Meeting of The
Japan Society for Bioscience, Biotechnology, andAgrochemistry, 3C03p15 (2020).
* 05tc16@gmail.com