Biotin sulfoxide reductase: Tryptophan 90 is required for efficient substrate utilization.
Rhodobacter sphaeroides f. sp. denitrificans biotin sulfoxide reductase (BSOR) catalyzes the reduction of d-biotin d-sulfoxide to biotin and contains the molybdopterin guanine dinucleotide (MGD) cofactor as its sole prosthetic group. Comparison of the primary sequences of BSOR and the closely related enzyme dimethyl sulfoxide reductase (DMSOR) indicated a number of conserved residues, including an active-site tryptophan residue (W90), which has been suggested to be involved in hydrogen bonding to the oxo group on the Mo(VI) center in BSOR. Site-directed mutagenesis has been used to replace tryptophan 90 in BSOR with phenylalanine, tyrosine, and alanine residues to examine the role of this residue in catalysis. All three BSOR mutant proteins were purified to homogeneity and contained MGD. The mutant proteins retained very limited activity toward the oxidizing substrates tested, with W90F retaining the most activity (3.4% of wild type). All three W90 mutant proteins exhibited greatly reduced k(cat) values compared to that of the wild-type enzyme, which was accompanied by little change in K(mapp). In addition, the mutant proteins had perturbed visible absorption and circular dichroism spectra suggesting different oxidation states of the Mo center. Purified samples of wild-type BSOR did not exhibit electron paramagnetic resonance (EPR) signals indicating a Mo(VI) center. After redox-cycling, partially reduced samples of wild-type BSOR revealed a proton-split S=1/2 Mo(V) resonance (g(1,2,3)=1.999, 1.981, 1.967; A(1,2,3)=1.40, 1.00, 1.05 mT) analogous to that observed in DMSOR. In contrast, EPR studies of the purified W90 mutant proteins revealed distinct S=1/2 Mo(V) resonances that were resistant to both oxidation and reduction, indicating that the Mo was trapped in the intermediate Mo(V) oxidation state. These results strongly suggest that W90 in BSOR plays a critical role in catalysis by serving as a hydrogen bond donor to the oxo group on the Mo(VI) center.
Pollock
Veronica V
VV
Department of Biochemistry and Molecular Biology, College of Medicine, University of South Florida, Tampa, FL 33612, USA.
Conover
Richard C
RC
Johnson
Michael K
MK
Barber
Michael J
MJ
D000409:Alanine / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D006150:Guanine Nucleotides / Q000737:chemistry / Q000378:metabolism*; D007506:Iron-Sulfur Proteins; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008982:Molybdenum / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000378:metabolism*; D010649:Phenylalanine / Q000737:chemistry; D011622:Pterins / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012242:Rhodobacter sphaeroides / Q000201:enzymology; D014364:Tryptophan / Q000737:chemistry*; D014443:Tyrosine / Q000737:chemistry
D006150:Guanine Nucleotides; D007506:Iron-Sulfur Proteins; D007527:Isoenzymes; D011622:Pterins; D011993:Recombinant Fusion Proteins; C063761:molybdopterin guanine dinucleotide; D014443:Tyrosine; D010649:Phenylalanine; D008982:Molybdenum; D014364:Tryptophan; D010088:Oxidoreductases; C019792:biotin sulfoxide reductase; C045664:dimethyl sulfoxide reductase; D000409:Alanine