Characterization of a thermostable NADPH:FMN oxidoreductase from the mesophilic bacterium Bacillus subtilis.


Abstract

The gene yhdA from Bacillus subtilis encoding a putative flavin mononucleotide (FMN)-dependent oxidoreductase was cloned and heterologously expressed in Escherichia coli. The purified enzyme has a noncovalently bound FMN cofactor, which is preferentially reduced by NADPH, indicating that YhdA is a NADPH:FMN oxidoreductase. The rate of NADPH oxidation is enhanced by the addition of external FMN, and analysis of initial rate measurements reveals the occurrence of a ternary complex in a bi-bi reaction mechanism. YhdA has also been shown to reductively cleave the -N=N- bond in azo dyes at the expense of NADPH, and hence, it possesses azoreductase activity, however, at a rate 100 times slower than that found for FMN. Using Cibacron Marine as a model compound, we could demonstrate that the dye is a competitive inhibitor of NADPH and FMN. The utilization of NADPH and the absence of a flavin semiquinone radical distinguish YhdA from flavodoxins, which adopt the same structural fold, i.e., a five-stranded beta sheet sandwiched by five alpha helices. The native molecular-mass of YhdA was determined to be 76 kDa, suggesting that the protein occurs as a tetramer, whereas the YhdA homologue in Saccharomyces cerevisiae (YLR011wp) forms a dimer in solution. Interestingly, the different oligomerization of these homologous proteins correlates to their thermostability, with YhdA exhibiting a melting point of 86.5 degrees C, which is 26.3 degrees C higher than that for the yeast protein. This unusually high melting point is proposed to be the result of increased hydrophobic packing between dimers and the additional presence of four salt bridges stabilizing the dimer-dimer interface. Study holds ProTherm entries: 20477, 20478 Extra Details: YhdA; azoreductase activity; hydrophobic packing; dimer-dimer interface

Submission Details

ID: gEgZWsfG

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:52 p.m.

Version: 1

Publication Details
Deller S;Sollner S;Trenker-El-Toukhy R;Jelesarov I;G├╝bitz GM;Macheroux P,Biochemistry (2006) Characterization of a thermostable NADPH:FMN oxidoreductase from the mesophilic bacterium Bacillus subtilis. PMID:16752898
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
2B0H 2006-01-17 Solution structure of VBS3 fragment of talin
2KC1 2010-01-19 NMR structure of the F0 domain (residues 0-85) of the talin ferm domain
2L7N 2011-12-14 NMR Structure of the B domain of talin
2L10 2011-08-31 Structure of the talin rod residues, domain C
6BA6 2017-12-06 Solution structure of Rap1b/talin complex
2KC2 2010-01-19 NMR structure of the F1 domain (residues 86-202) of the talin
5O2Q 2017-09-20 p130Cas SH3 domain Vinculin peptide chimera
2KGX 2009-03-31 HADDOCK structure of the talin F3 domain in complex with talin 1655-1822
2KVP 2010-05-05 NMR structure of the talin VBS3 domain, 1815-1973
2L7A 2011-12-21 Solution Structure of the talin VBS2b domain
2MWN 2014-12-17 Talin-F3 / RIAM N-terminal Peptide complex
2JSW 2008-01-29 NMR structure of the talin C-terminal actin binding site
2KMA 2010-03-02 NMR structure of the F0F1 double domain (residues 1-202) of the talin ferm domain
2G35 2006-05-02 NMR structure of talin-PTB in complex with PIPKI
2KBB 2009-03-17 NMR structure of the talin rod domain, 1655-1822
1U89 2005-01-18 Solution structure of VBS2 fragment of talin
2LQG 2013-02-13 Solution Structure of the R4 domain of talin
4LN2 2014-05-28 1.0 The second SH3 domain from CAP/Ponsin in complex with proline rich peptide from Vinculin
4LNP 2014-05-28 1.41 The first SH3 domain from CAP/Ponsin in complex with proline rich peptide from Vinculin
4W8P 2014-12-03 1.5 Crystal structure of RIAM TBS1 in complex with talin R7R8 domains
3RF3 2011-04-27 1.61 Shigella IpaA-VBS3 in complex with human vinculin
1YDI 2005-07-19 1.8 Human Vinculin Head Domain (VH1, 1-258) in Complex with Human Alpha-Actinin's Vinculin-Binding Site (Residues 731-760)
1QKR 2000-08-04 1.8 Crystal structure of the vinculin tail and a pathway for activation
1ZVZ 2005-09-20 1.8 Vinculin Head (0-258) in Complex with the Talin Rod Residue 820-844
3DYJ 2009-01-27 1.85 Crystal structure a talin rod fragment
3IVF 2010-08-18 1.94 Crystal structure of the talin head FERM domain
3S90 2011-06-22 1.97 Human vinculin head domain Vh1 (residues 1-252) in complex with murine talin (VBS33; residues 1512-1546)
5IC0 2016-05-18 1.97 Structural analysis of a talin triple domain module
3TJ5 2011-09-07 1.99 human vinculin head domain (Vh1, residues 1-258) in complex with the vinculin binding site of the surface cell antigen 4 (sca4-VBS-N; residues 412-434) from Rickettsia rickettsii
2X0C 2010-07-07 2.0 STRUCTURE OF THE TALIN ROD RESIDUES 1359-1659
1T0I 2004-06-22 2.0 YLR011wp, a Saccharomyces cerevisiae NA(D)PH-dependent FMN reductase
4F7G 2012-07-04 2.05 Crystal structure of talin autoinhibition complex
1T01 2004-08-24 2.06 Vinculin complexed with the VBS1 helix from talin
1ZW2 2005-09-20 2.1 Vinculin Head (0-258) in Complex with the Talin Rod residues 2345-2369
5FZT 2016-04-27 2.1 The crystal structure of R7R8 in complex with a DLC1 fragment.
3GFS 2009-09-22 2.11 Structure of YhdA, K109D/D137K variant
2QDQ 2008-01-29 2.2 Crystal structure of the talin dimerisation domain
3MYI 2010-05-26 2.2 Human metavinculin tail domain
5IC1 2016-05-18 2.2 Structural analysis of a talin triple domain module, E1794Y, E1797Y, Q1801Y mutant
4DJ9 2012-02-29 2.25 Human vinculin head domain Vh1 (residues 1-258) in complex with the talin vinculin binding site 50 (VBS50, residues 2078-2099)
4E17 2012-05-16 2.3 Alpha-E-catenin is an autoinhibited molecule that co-activates vinculin
3ZDL 2013-02-13 2.3 Vinculin head (1-258) in complex with a RIAM fragment
1RKE 2004-01-13 2.35 Human vinculin head (1-258) in complex with human vinculin tail (879-1066)
1U6H 2005-01-18 2.38 Vinculin head (0-258) in complex with the talin vinculin binding site 2 (849-879)
3GFR 2009-09-22 2.4 Structure of YhdA, D137L variant
4E18 2012-05-16 2.4 Alpha-E-catenin is an autoinhibited molecule that co-activates vinculin
1SYQ 2004-07-20 2.42 Human vinculin head domain VH1, residues 1-258, in complex with human talin's vinculin binding site 1, residues 607-636
5L0I 2016-08-31 2.45 Human metavinculin MVt R975W cardiomyopathy-associated mutant (residues 959-1134)
5NL1 2018-05-16 2.5 Shigella IpaA-VBS3/TBS in complex with the Talin VBS1 domain 488-512
1SJ7 2004-08-24 2.5 Crystal Structure of Talin Rod 482-655
1NNI 2003-07-29 2.5 Azobenzene Reductase from Bacillus subtilis
3VF0 2012-07-25 2.54 Raver1 in complex with metavinculin L954 deletion mutant
1Y19 2005-01-04 2.6 Structural basis for phosphatidylinositol phosphate kinase type I-gamma binding to talin at focal adhesions
1XWJ 2005-10-11 2.6 Vinculin head (1-258) in complex with the talin vinculin binding site 3 (1945-1969)
1SJ8 2004-08-24 2.6 Crystal Structure of talin residues 482-789
4EHP 2012-04-18 2.66 Crystal Structure of human vinculin head domain (residues 1-252) in complex with alpha-catenin (residues 277-382)
1RKC 2004-01-13 2.7 Human vinculin head (1-258) in complex with talin's vinculin binding site 3 (residues 1944-1969)
2GWW 2006-11-14 2.72 Human vinculin (head domain, Vh1, residues 1-258) in complex with Shigella's IpaA vinculin binding site (residues 602-633)
2GDC 2006-08-08 2.74 Structure of Vinculin VD1 / IpaA560-633 complex
5L0D 2016-08-31 2.75 Human Metavinculin(residues 959-1130) in complex with PIP2
3H2U 2009-07-28 2.75 Human raver1 RRM1, RRM2, and RRM3 domains in complex with human vinculin tail domain Vt
5L0F 2016-08-31 2.76 Human metavinculin quadruple mutant (residues 959-1134)
3TJ6 2011-09-07 2.76 human vinculin head domain (Vh1, residues 1-258) in complex with the vinculin binding site of the surface cell antigen 4 (sca4-VBS-C; residues 812-835) from Rickettsia rickettsii
5Y04 2018-03-28 2.85 Crystal Structure of the complex between the vinculin D1 domain and alphaE-catenin
6MFS 2018-09-26 2.85 Mouse talin1 residues 1-138 fused to residues 169-400 in complex with phosphatidylinositol 4,5-bisphosphate (PIP2)
6FQ4 2018-05-09 2.89 Structure of Chlamydial virulence factor TarP and vinculin head domain
3H2V 2009-07-28 2.9 Human raver1 RRM1 domain in complex with human vinculin tail domain Vt
1TR2 2005-11-15 2.9 Crystal structure of human full-length vinculin (residues 1-1066)
5L0H 2016-08-31 2.9 Human metavinculin MVt cardiomyopathy-associated mutant R975W (residues 959-1134) in complex with PIP2
2GSW 2006-05-09 2.92 Crystal Structure of the Putative NADPH-dependent Azobenzene FMN-Reductase YhdA from Bacillus subtilis, Northeast Structural Genomics Target SR135
3GFQ 2009-09-22 3.0 Structure of YhdA, K109L variant
6FUY 2018-03-14 3.0 Crystal structure of human full-length vinculin-T12-A974K (residues 1-1066)
5L0C 2016-08-31 3.1 Human metavinculin (residues 959-1134) in complex with PIP2
1ST6 2004-08-03 3.1 Crystal structure of a cytoskeletal protein
4PR9 2014-12-10 3.2 Human Vinculin (residues 891-1066) in complex with PIP
2IBF 2007-09-18 3.2 Human vinculin's head domain (Vh1, residues 1-258) in complex with two vinculin binding sites of Shigella flexneri's IpaA (residues 565-587)
1ZW3 2005-09-20 3.3 Vinculin Head (0-258) in Complex with the Talin Rod residues 1630-1652
5L0G 2016-08-31 3.4 Human metavinculin MVt Q971R, R975D, T978R mutant (residues 959-1134) in complex with PIP2
2HSQ 2006-11-21 3.97 Human vinculin (head domain, Vh1, residues 1-258) in complex with Shigella's IpaA vinculin binding site 2 (residues 565-587)
5L0J 2016-08-31 4.0 Human vinculin R903Q, D907R, R910T mutant(residues 891-1066)
3JBK 2015-11-04 8.2 Cryo-EM reconstruction of the metavinculin-actin interface
3JBI 2015-11-04 8.5 MDFF model of the vinculin tail domain bound to F-actin

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 NAD(P)H-dependent FMN reductase LOT6 Q07923 LOT6_YEAST
100.0 FMN-dependent NADPH-azoreductase O07529 AZR_BACSU
100.0 B Vinculin P26039 TLN1_MOUSE
100.0 B Vinculin Q9Y490 TLN1_HUMAN
97.6 A Vinculin P85972 VINC_RAT
98.0 A Vinculin P26234 VINC_PIG
98.0 A Vinculin Q64727 VINC_MOUSE
98.0 A Vinculin P18206 VINC_HUMAN
100.0 A Vinculin P12003 VINC_CHICK