Trp(359) regulates flavin thermodynamics and coenzyme selectivity in Mycobacterium tuberculosis FprA.
Mtb (Mycobacterium tuberculosis) FprA (flavoprotein reductase A) is an NAD(P)H-dependent FAD-binding reductase that is structurally related to mammalian adrenodoxin reductase, and which supports the catalytic function of Mtb cytochrome P450s. Trp(359), proximal to the FAD, was investigated in light of its potential role in controlling coenzyme interactions, as observed for similarly located aromatic residues in diflavin reductases. Phylogenetic analysis indicated that a tryptophan residue corresponding to Trp(359) is conserved across FprA-type enzymes and in adrenodoxin reductases. W359A/H mutants of Mtb FprA were generated, expressed and the proteins characterized to define the role of Trp(359). W359A/H mutants exhibited perturbed UV-visible absorption/fluorescence properties. The FAD semiquinone formed in wild-type NADPH-reduced FprA was destabilized in the W359A/H mutants, which also had more positive FAD midpoint reduction potentials (-168/-181 mV respectively, versus the standard hydrogen electrode, compared with -230 mV for wild-type FprA). The W359A/H mutants had lower ferricyanide reductase k(cat) and NAD(P)H K(m) values, but this led to improvements in catalytic efficiency (k(cat)/K(m)) with NADH as reducing coenzyme (9.6/18.8 muM(-1).min(-1) respectively, compared with 5.7 muM(-1).min(-1) for wild-type FprA). Stopped-flow spectroscopy revealed NAD(P)H-dependent FAD reduction as rate-limiting in steady-state catalysis, and to be retarded in mutants (e.g. limiting rate constants for NADH-dependent FAD reduction were 25.4 s(-1) for wild-type FprA and 4.8 s(-1)/13.4 s(-1) for W359A/H mutants). Diminished mutant FAD content (particularly in W359H FprA) highlighted the importance of Trp(359) for flavin stability. The results demonstrate that the conserved Trp(359) is critical in regulating FprA FAD binding, thermodynamic properties, catalytic efficiency and coenzyme selectivity.
Neeli
Rajasekhar
R
Manchester Interdisciplinary Biocentre, The University of Manchester, Faculty of Life Sciences, 131 Princess Street, Manchester M1 7DN, UK.
Sabri
Muna
M
McLean
Kirsty J
KJ
Dunford
Adrian J
AJ
Scrutton
Nigel S
NS
Leys
David
D
Munro
Andrew W
AW
D000595:Amino Acid Sequence; D000818:Animals; D003067:Coenzymes / Q000737:chemistry* / Q000378:metabolism*; D017124:Conserved Sequence; D005415:Flavins / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013053:Spectrophotometry; D013816:Thermodynamics; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism
D003067:Coenzymes; D005415:Flavins; D014364:Tryptophan; C464438:FprA protein, M tuberculosis; D009247:NADH, NADPH Oxidoreductases