Crystal structures of the short-chain flavin reductase HpaC from Sulfolobus tokodaii strain 7 in its three states: NAD(P)(+)(-)free, NAD(+)(-)bound, and NADP(+)(-)bound.
4-Hydroxyphenylacetate (4-HPA) is oxidized as an energy source by two component enzymes, the large component (HpaB) and the small component (HpaC). HpaB is a 4-HPA monooxygenase that utilizes FADH(2) supplied by a flavin reductase HpaC. We determined the crystal structure of HpaC (ST0723) from the aerobic thermoacidophilic crenarchaeon Sulfolobus tokodaii strain 7 in its three states [NAD(P)(+)-free, NAD(+)-bound, and NADP(+)-bound]. HpaC exists as a homodimer, and each monomer was found to contain an FMN. HpaC preferred FMN to FAD because there was not enough space to accommodate the AMP moiety of FAD in its flavin-binding site. The most striking difference between the NAD(P)(+)-free and the NAD(+)/NADP(+)-bound structures was observed in the N-terminal helix. The N-terminal helices in the NAD(+)/NADP(+)-bound structures rotated ca. 20 degrees relative to the NAD(P)(+)-free structure. The bound NAD(+) has a compact folded conformation with nearly parallel stacking rings of nicotinamide and adenine. The nicotinamide of NAD(+) stacked the isoalloxazine ring of FMN so that NADH could directly transfer hydride. The bound NADP(+) also had a compact conformation but was bound in a reverse direction, which was not suitable for hydride transfer.
Okai
Masahiko
M
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Kudo
Norio
N
Lee
Woo Cheol
WC
Kamo
Masayuki
M
Nagata
Koji
K
Tanokura
Masaru
M
D000595:Amino Acid Sequence; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D038181:FMN Reductase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006860:Hydrogen Bonding; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009243:NAD / Q000737:chemistry* / Q000378:metabolism*; D009249:NADP / Q000737:chemistry* / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D016944:Sulfolobus / Q000201:enzymology* / Q000235:genetics
D021122:Protein Subunits; D009243:NAD; D009249:NADP; D038181:FMN Reductase