SASDDP3: N-propargyl glycine-Inactivated Proline utilization A from Bradyr...

Basic information

• Entry: Database: SASBDB / ID: SASDDP3

Sample

N-propargyl glycine-Inactivated Proline utilization A from Bradyrhizobium diazoefficiens (formerly Bradyrhizobium japonicum) collected by SEC-SAXS

• Bifunctional protein PutA (protein), BjPutA, Bradyrhizobium diazoefficiens (strain JCM 10833 / IAM 13628 / NBRC 14792 / USDA 110)

Function / homology

Function:

proline dehydrogenase / proline dehydrogenase activity / 1-pyrroline-5-carboxylate dehydrogenase activity / L-glutamate gamma-semialdehyde dehydrogenase / proline catabolic process to glutamate / proline biosynthetic process / cytoplasmic side of plasma membrane / DNA-binding transcription factor activity / nucleotide binding / DNA binding / identical protein binding

Domain/homology:

Delta-1-pyrroline-5-carboxylate dehydrogenase 3 / Proline dehydrogenase PutA, domain II / Proline dehydrogenase PutA, domain I/II / DNA-binding domain of Proline dehydrogenase / Bifunctional protein PutA / Proline dehydrogenase domain / Proline dehydrogenase / FAD-linked oxidoreductase-like / Aldehyde dehydrogenase, cysteine active site / Aldehyde dehydrogenases cysteine active site. / Aldehyde dehydrogenase domain / Aldehyde dehydrogenase family / Aldehyde dehydrogenase, N-terminal / Aldehyde dehydrogenase, C-terminal / Aldehyde/histidinol dehydrogenase

Component:

Bifunctional protein PutA

• Biological species: Bradyrhizobium diazoefficiens (strain JCM 10833 / IAM 13628 / NBRC 14792 / USDA 110) (bacteria)
• Citation: Journal: Biophys J / Year: 2018; Title: Redox Modulation of Oligomeric State in Proline Utilization A.; Authors: David A Korasick / Ashley C Campbell / Shelbi L Christgen / Srinivas Chakravarthy / Tommi A White / Donald F Becker / John J Tanner / ; Abstract: Homooligomerization of proline utilization A (PutA) bifunctional flavoenzymes is intimately tied to catalytic function and substrate channeling. PutA from Bradyrhizobium japonicum (BjPutA) is unique among PutAs in that it forms a tetramer in solution. Curiously, a dimeric BjPutA hot spot mutant was previously shown to display wild-type catalytic activity despite lacking the tetrameric structure. These observations raised the question of what is the active oligomeric state of BjPutA. Herein, we investigate the factors that contribute to tetramerization of BjPutA in vitro. Negative-stain electron microscopy indicates that BjPutA is primarily dimeric at nanomolar concentrations, suggesting concentration-dependent tetramerization. Further, sedimentation-velocity analysis of BjPutA at high (micromolar) concentration reveals that although the binding of active-site ligands does not alter oligomeric state, reduction of the flavin adenine dinucleotide cofactor results in dimeric protein. Size-exclusion chromatography coupled with multiangle light scattering and small-angle x-ray scattering analysis also reveals that reduced BjPutA is dimeric. Taken together, these results suggest that the BjPutA oligomeric state is dependent upon both enzyme concentration and the redox state of the flavin cofactor. This is the first report, to our knowledge, of redox-linked oligomerization in the PutA family.

Contact author

• David Korasick (Mizzou, University of Missouri-Columbia, Columbia, MO, USA)

Models

• Model #1805: ; Type: atomic / Chi-square value: 2.9816132533; Search similar-shape structures of this assembly by Omokage search (details)
• Model #1795: ; Type: atomic / Chi-square value: 1.64908802698; Search similar-shape structures of this assembly by Omokage search (details)
• Model #1796: ; Type: atomic / Chi-square value: 1.64908802698; Search similar-shape structures of this assembly by Omokage search (details)

Sample

• Sample: Name: N-propargyl glycine-Inactivated Proline utilization A from Bradyrhizobium diazoefficiens (formerly Bradyrhizobium japonicum) collected by SEC-SAXS
• Buffer: Name: 50 mM Tris, 50 mM NaCl, 0.5 mM TCEP, 5% (v/v) glycerol; pH: 7.8

Entity #976

Name: BjPutA / Type: protein / Description: Bifunctional protein PutA / Formula weight: 107.558 / Num. of mol.: 2
Source: Bradyrhizobium diazoefficiens (strain JCM 10833 / IAM 13628 / NBRC 14792 / USDA 110)
References: UniProt: Q89E26
Sequence:

GHMPNIPPPF TAPYAPDDAE IAARLLPASH LSPPQEARIH RTATRLIEAI RKRDDRLGGV EDMLREFALS TKEGLALMVL AEALLRVPDA RTADQFIEDK LGEGDFIHHE TKSTAFLVNA SAWALGLSAR VIQPGETPDG TIGRLVKRLG APAVRTATRQ AMRLMGNHFV LGETIEQALE RGKPRSGQKT RYSFDMLGEG ARTAADARRY FDAYASAIET IGKAAGNHAL PDRPGISVKL SALHPRFEAI SRARVMVELV PQLLDLAQRA KAHDLNFTVD AEEADRLELS LDVIAATLAD PSLKGWDGFG LAIQAYQKRA SAVIDYVDAL ARAHDRKLMV RLVKGAYWDT EIKRAQERGL DGYPVFTRKA MTDLNYVACA SKLLALRPRI FPQFATHNAL TVATVLEMAE GSSGFEFQRL HGMGEALYEQ LAKDHADIAY RTYAPVGSHR DLLAYLVRRL LENGANSSFV AQAADYRVPV PALLQRPADA IVRPQAAAHP RIPLPCDLFA PERRNSRGVE FGARTALDQL LTDVKAETGD LKPIADATPD QAHAAVAAAR AGFAGWSRTP AGIRAAALEQ AAHLLESRSA HFIALLQREG GKTLDDALSE LREAADFCRY YAAQGRKLFG SETAMPGPTG ESNALTMRGR GVFVAISPWN FPLAIFLGQV TAALMAGNSV VAKPAEQTPR IAREAVALLH EAGIPKSALY LVTGDGRIGA ALTAHPDIAG VVFTGSTEVA RSINRALAAK DGPIVPLIAE TGGINAMIAD ATALPEQVAD DVVTSAFRSA GQRCSALRLL FVQEDVADRM IEMVAGAARE LKIGDPSDVA THVGPVIDVE AKQRLDAHIA RMKTEARLHF AGPAPEGCFV APHIFELTEA GQLTEEVFGP ILHVVRYRPE NLERVLRAIE RTGYGLTLGV HSRIDDSIEA IIDRVQVGNI YVNRNMIGAV VGVQPFGGNG LSGTGPKAGG PHYLARFATE QTVTINTAAA GGNAALLAGE E

Experimental information

• Beam: Instrument name: Advanced Photon Source (APS) BioCAT 18ID / City: Argonne, IL / 国: USA / Type of source: X-ray synchrotronSynchrotron / Wavelength: 0.103 Å / Dist. spec. to detc.: 3.5 mm
• Detector: Name: Pilatus 100K / Pixsize x: 172 mm

Scan

Title: N-propargyl glycine-Inactivated Proline utilization A from Bradyrhizobium diazoefficiens (formerly Bradyrhizobium japonicum) collected by SEC-SAXS
Measurement date: Jul 16, 2017 / Cell temperature: 22 °C / Unit: 1/A /

	Min	Max
Q	0.0057	0.3826

Distance distribution function P(R)

Sofotware P(R): GNOM 5.0 / Number of points: 1264 /

	Min	Max
Q	0.00569995	0.382606
P(R) point	1	1264
R	0	144.2

Result

Type of curve: sec /

	Experimental	Porod
MW	249.7 kDa	-
Volume	-	324 nm3

	P(R)	Guinier	Guinier error
Forward scattering, I0	380.4	377.5	0.9
Radius of gyration, Rg	4.7 nm	4.63 nm	0.02

	Min	Max
D	-	14.42
Guinier point	1	76