SASDCQ3: Proline utilization A from Desulfovibrio vulgaris 1.5 mg/mL

Basic information

Entry

Database: SASBDB / ID: SASDCQ3

Sample

Proline utilization A from Desulfovibrio vulgaris 1.5 mg/mL

• Bifunctional protein PutA (protein), DvPutA, Desulfovibrio vulgaris (strain RCH1)

Function / homology

Function:

proline dehydrogenase / proline dehydrogenase activity / 1-pyrroline-5-carboxylate dehydrogenase activity / L-glutamate gamma-semialdehyde dehydrogenase / proline catabolic process to glutamate / oxidoreductase activity, acting on the aldehyde or oxo group of donors, NAD or NADP as acceptor / proline biosynthetic process / DNA-binding transcription factor activity / DNA binding

Domain/homology:

Proline utilization A, N-terminal / Proline utilization A N-terminal domain / 1-pyrroline-5-carboxylate dehydrogenase / Bifunctional protein PutA / Proline dehydrogenase domain / Proline dehydrogenase / FAD-linked oxidoreductase-like / Aldehyde dehydrogenase, glutamic acid active site / Aldehyde dehydrogenases glutamic acid active site. / 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: Desulfovibrio vulgaris (strain RCH1) (bacteria)
• Citation: Journal: FEBS J / Year: 2017; Title: Biophysical investigation of type A PutAs reveals a conserved core oligomeric structure.; Authors: David A Korasick / Harkewal Singh / Travis A Pemberton / Min Luo / Richa Dhatwalia / John J Tanner / ; Abstract: Many enzymes form homooligomers, yet the functional significance of self-association is seldom obvious. Herein, we examine the connection between oligomerization and catalytic function for proline utilization A (PutA) enzymes. PutAs are bifunctional enzymes that catalyze both reactions of proline catabolism. Type A PutAs are the smallest members of the family, possessing a minimal domain architecture consisting of N-terminal proline dehydrogenase and C-terminal l-glutamate-γ-semialdehyde dehydrogenase modules. Type A PutAs form domain-swapped dimers, and in one case (Bradyrhizobium japonicum PutA), two of the dimers assemble into a ring-shaped tetramer. Whereas the dimer has a clear role in substrate channeling, the functional significance of the tetramer is unknown. To address this question, we performed structural studies of four-type A PutAs from two clades of the PutA tree. The crystal structure of Bdellovibrio bacteriovorus PutA covalently inactivated by N-propargylglycine revealed a fold and substrate-channeling tunnel similar to other PutAs. Small-angle X-ray scattering (SAXS) and analytical ultracentrifugation indicated that Bdellovibrio PutA is dimeric in solution, in contrast to the prediction from crystal packing of a stable tetrameric assembly. SAXS studies of two other type A PutAs from separate clades also suggested that the dimer predominates in solution. To assess whether the tetramer of B. japonicum PutA is necessary for catalytic function, a hot spot disruption mutant that cleanly produces dimeric protein was generated. The dimeric variant exhibited kinetic parameters similar to the wild-type enzyme. These results implicate the domain-swapped dimer as the core structural and functional unit of type A PutAs.; ENZYMES: Proline dehydrogenase (EC 1.5.5.2); l-glutamate-γ-semialdehyde dehydrogenase (EC 1.2.1.88).; DATABASES: The atomic coordinates and structure factor amplitudes have been deposited in the Protein Data Bank under accession number 5UR2. The SAXS data have been deposited in the SASBDB under the following accession codes: SASDCP3 (BbPutA), SASDCQ3 (DvPutA 1.5 mg·mL ), SASDCX3 (DvPutA 3.0 mg·mL ), SASDCY3 (DvPutA 4.5 mg·mL ), SASDCR3 (LpPutA 3.0 mg·mL ), SASDCV3 (LpPutA 5.0 mg·mL ), SASDCW3 (LpPutA 8.0 mg·mL ), SASDCS3 (BjPutA 2.3 mg·mL ), SASDCT3 (BjPutA 4.7 mg·mL ), SASDCU3 (BjPutA 7.0 mg·mL ), SASDCZ3 (R51E 2.3 mg·mL ), SASDC24 (R51E 4.7 mg·mL ), SASDC34 (R51E 7.0 mg·mL ).

Contact author

• John Tanner (Mizzou, University of Missouri-Columbia, Columbia, MO, USA)

Models

Sample

• Sample: Name: Proline utilization A from Desulfovibrio vulgaris 1.5 mg/mL; Specimen concentration: 1.5 mg/ml
• Buffer: Name: 50 mM Tris-HCl, 50 mM NaCl, 0.5 mM EDTA, and 0.5 mM THP at pH 7.5.; pH: 7.5

Entity #679

Name: DvPutA / Type: protein / Description: Bifunctional protein PutA / Formula weight: 114.496 / Num. of mol.: 2 / Source: Desulfovibrio vulgaris (strain RCH1) / References: UniProt: A0A0E0T6R2
Sequence:

MHHHHHHSSG VDLGTENLYF QSMDQQHLDG KVVERGKEFF RSISGEAPSI FNKGWWTGKV MDWAMQNEDF KVQLFRFVDV LPYLNTSESL LRHIREYFAT EDADIPPVLK WGAGKAGIGG ALTAKLMGMT IRSNIEGMAR QFIIGDNSKE AVKGLAKLRK DGFTFTVDLL GEATVSEEES EAYAQGYHEV VDAIAREQEK WKALPGNGPV EGFDWGATPK VNVSIKPSAL YSQAKPVDVE GSVRGILSRL VPIYRKVVAM GGFLCIDMEQ LKYKEMTLEL FKRLRSDPEF RHYPHLSIVL QAYLRDTEKD LDDLLHWARS EKLPIGIRLV KGAYWDYETV IAKQNGWEIP VWTDKPESDI AYEKLAHRIL ENSDIVYFAC ASHNVRTIAA VMETALALNV PEHRYEFQVL YGMAEPVRKG LKNVAGRVRL YCPYGELIPG MAYLVRRLLE NTANESFLRQ SFAEGAALER LLENPQKTLH RLLAARPEPR AVEPGPGGLP PFTNDAMIDF TVPDNRKAFV EALADVRSRF GQTVPLYIGG RDVTTADLIP TTNPAKPAEV VASICQAGRP EIDDAIAAAK KAALTWRDTS PADRAAYLRR AADICRKRIW ELSAWQVVEV GKQWDQAYHD VTEGIDFLEY YAREMLRLGA PRRMGRAPGE HNHLFYQPKG IAAVIAPWNF PFAIAIGMAS AAIVTGNPVI FKPSSISSRI GYNLAEVFRE AGLPEGVFNY CPGRSSIMGD YLVEHPDISL ICFTGSMEVG LRIQEKAAKV QPGQRQCKRV IAEMGGKNAT IIDDDADLDE AVLQVLYSAF GFQGQKCSAC SRVIVLDAIY DRFIERLVKA ASSIHIGPSE DPSNYMGPVA DATLQKNVSD YIRIAEEEGR VLLKRTDLPA EGCYVPLTIV GDIRPEHRIA QEEIFGPVLA VMRAATFDEA LSIANGTRFA LTGAVFSRSP EHLDKARREF RVGNLYLNKG STGALVERQP FGGFAMSGVG SKTGGPDYLL QFMDPRVVTE NTMRRGFTPI DEDDDWIV

Experimental information

• Beam: Instrument name: Advanced Light Source (ALS) 12.3.1 (SIBYLS); City: Berkeley, CA / 国: USA / Type of source: X-ray synchrotronSynchrotron
• Detector: Name: MAR 165 CCD

Scan

Title: Proline utilization A from Desulfovibrio vulgaris 1.5 mg/mL
Measurement date: Jun 8, 2012 / Unit: 1/A /

	Min	Max
Q	0.0125	0.3214

Distance distribution function P(R)

Sofotware P(R): GNOM 5.0 / Number of points: 509 /

	Min	Max
Q	0.012467	0.321359
P(R) point	1	509
R	0	160

Result

Type of curve: single_conc /

	Experimental	Porod
MW	213 kDa	-
Volume	-	293 nm3

	P(R)	P(R) error	Guinier	Guinier error
Forward scattering, I0	702	3.4	681.48	4.03
Radius of gyration, Rg	4.67 nm	0.02	4.36 nm	0.04

	Min	Max
D	-	16
Guinier point	1	29