Mutagenesis of Glycine 179 Modulates Both Catalytic Efficiency and Reduced Pyridine Nucleotide Specificity in Cytochrome b₅ Reductase

Abstract

Cytochrome b5 reductase (cb5r), a member of the ferredoxin:NADP+ reductase family of flavoprotein transhydrogenases, catalyzes the NADH-dependent reduction of cytochrome b5. Within this family, a conserved "GxGxxP" sequence motif has been implicated in binding reduced pyridine nucleotides. However, Glycine 179, a conserved residue in cb5r primary structures, precedes this six-residue "180GxGxxP185" motif that has been identified as binding the adenosine moiety of NADH. To investigate the role of G179 in NADH complex formation and NAD(P)H specificity, a series of rat cb5r variants were generated, corresponding to G179A, G179P, G179T, and G179V, recombinantly expressed in Escherichia coli and purified to homogeneity. Each mutant protein was found to incorporate FAD in a 1:1 cofactor/protein stoichiometry and exhibited absorption and CD spectra that were identical to those of wild-type cb5r, indicating both correct protein folding and similar flavin environments, while oxidation-reduction potentials for the FAD/FADH2 couple (n = 2) were also comparable to the wild-type protein (E(o)' = -272 mV). All four mutants showed decreased NADH:ferricyanide reductase activities, with kcat decreasing in the order WT > G179A > G179P > G179T > G179V, with the G179V variant retaining only 1.5% of the wild-type activity. The affinity for NADH also decreased in the order WT > G179A > G179P > G179T > G179V, with the Km(NADH) for G179V 180-fold greater than that of the wild type. Both Ks(H4NAD) and Ks(NAD+) values confirmed that the G179 mutants had both compromised NADH- and NAD+-binding affinities. Determination of the NADH/NADPH specificity constant for the various mutants indicated that G179 also participated in pyridine nucleotide selectivity, with the G179V variant preferring NADPH approximately 8000 times more than wild-type cb5r. These results demonstrated that, while G179 was not critical for either flavin incorporation or maintenance of the appropriate flavin environment in cb5r, G179 was required for both effective NADH/NADPH selectivity and to maintain the correct orientation and position of the conserved cysteine in the proline-rich "CGpppM" motif that is critical for optimum NADH binding and efficient hydride transfer.

Characterization of the CDP-2-Glycerol Biosynthetic Pathway in Streptococcus pneumoniae

“…Compared to the above dehydrogenases, Gtp1 was identified as a reductase catalyzing the conversion of 1,3-dihydroxyacetone or glyceraldehyde to glycerol and as requiring NADH/NADPH as a cofactor; it was not active for the reverse reaction, and Fe 2ϩ instead of Zn 2ϩ was favorable for Gtp1 activity. Several NADH/NADPH binding motifs, such as GXGXXA (24), GXGXXG (23), and GX GXXP (21), have been reported, and we proposed that the GXGXXR motif near the position of the NAD ϩ binding site may be the possible motif for Gtp1 to combine with NADH/ NADPH. The binding sites of Zn 2ϩ in G1PDH may be responsible for the binding of Fe 2ϩ in Gtp2; however, further experimental evidence is needed.…”

A fragment of adhesion molecule L1 is imported into mitochondria, and regulates mitochondrial metabolism and trafficking

“…The Rossmann motif is part of the Rossmann fold, which is present in proteins that bind nucleotides, such as enzyme cofactors FMN, FAD, and NAD as well as ATP. In addition to the GxGxxG motif, other motifs, such as GxGxxP, GxxxG/A and RxS/ T (Dym and Eisenberg, 2001;Kleiger and Eisenberg, 2002;Roma et al, 2005), are involved in binding of NADH, NADPH, FAD or FMN. Interestingly, two GxGxxG motifs (G 794 KGPEP, G 899 RGSGP), five GxxxG motifs (G 842 HLRG, G 899 RGSG G 946 VLTG, G 1223 SFIG, G 1227 QYSG), five GxxxA motifs (G 918 HPEA, G 1000 PGEA, G 1009 GTMA, G 1021 NISA, G 1124 FVSA) and one RxYS motif (R 885 PYS) are present in L1-70, suggesting that L1-70 binds to nucleotides, such as NADH.…”

The Zebrafish Cytochrome b5/Cytochrome b5 Reductase/NADH System Efficiently Reduces Cytoglobins 1 and 2: Conserved Activity of Cytochrome b5/Cytochrome b5 Reductases during Vertebrate Evolution

“…Similar observations have been reported for other CYB5R proteins, where the one-electron reduced, semiquinone state is not stable and does not accumulate during the reduction step. 43,[47][48][49] Thus, the two one-electron transitions show a very similar midpoint potential and the two steps are merged in a single apparent E m value.…”