SASDB27: Chimeric EcRHH-RcPutA: The E.coli Proline utilization A RHH domai...

Basic information

Entry

Database: SASBDB / ID: SASDB27

Sample

Chimeric EcRHH-RcPutA: The E.coli Proline utilization A RHH domain fused to R.capsulatus PutA

• Proline utilization A (protein), PutA, Escherchia coli, Rhodobacter capsulatus

• Biological species: Escherchia coli (E. coli); Rhodobacter capsulatus (bacteria)
• Citation: Journal: Biosci Rep / Year: 2016; Title: Engineering a trifunctional proline utilization A chimaera by fusing a DNA-binding domain to a bifunctional PutA.; Authors: Benjamin W Arentson / Erin L Hayes / Weidong Zhu / Harkewal Singh / John J Tanner / Donald F Becker / ; Abstract: Proline utilization A (PutA) is a bifunctional flavoenzyme with proline dehydrogenase (PRODH) and Δ-pyrroline-5-carboxylate (P5C) dehydrogenase (P5CDH) domains that catalyses the two-step oxidation of proline to glutamate. Trifunctional PutAs also have an N-terminal ribbon-helix-helix (RHH) DNA-binding domain and moonlight as autogenous transcriptional repressors of the put regulon. A unique property of trifunctional PutA is the ability to switch functions from DNA-bound repressor to membrane-associated enzyme in response to cellular nutritional needs and proline availability. In the present study, we attempt to construct a trifunctional PutA by fusing the RHH domain of Escherichia coli PutA (EcRHH) to the bifunctional Rhodobacter capsulatus PutA (RcPutA) in order to explore the modular design of functional switching in trifunctional PutAs. The EcRHH-RcPutA chimaera retains the catalytic properties of RcPutA while acquiring the oligomeric state, quaternary structure and DNA-binding properties of EcPutA. Furthermore, the EcRHH-RcPutA chimaera exhibits proline-induced lipid association, which is a fundamental characteristic of functional switching. Unexpectedly, RcPutA lipid binding is also activated by proline, which shows for the first time that bifunctional PutAs exhibit a limited form of functional switching. Altogether, these results suggest that the C-terminal domain (CTD), which is conserved by trifunctional PutAs and certain bifunctional PutAs, is essential for functional switching in trifunctional PutAs.

Contact author

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

Models

Sample

• Sample: Name: Chimeric EcRHH-RcPutA: The E.coli Proline utilization A RHH domain fused to R.capsulatus PutA; Specimen concentration: 6.00-6.00
• Buffer: Name: 50 mM Tris, 200 mM NaCl, 0.5 mM Tris(3-hydroxypropyl)phosphine; pH: 7.5

Entity #429

Name: PutA / Type: protein / Description: Proline utilization A / Formula weight: 125.528 / Num. of mol.: 2 / Source: Escherchia coli, Rhodobacter capsulatus
Sequence:

MGSSHHHHHH SSGLVPRGSH MGTTTMGVKL DDATRERIKS AATRIDRTPH WLIKQAIFSY LEQLENSDTL PEHMTDLSAL GPKAKFAPEA EVLQALVAQA ALPQPQLDRI AARGADLVAR IRAEAKPSLM EHFLAQYGLS TREGVALMCL AEAMLRVPDT ATIDALIEDK IAPSDWGKHL GTAASSLVNA STWALMLTGK VLDDGAGGIA GTLRGAMRRL GEPVIRAAVG QAMREMGRQF VLGETIEKAL ERAEKREAEG YTFSYDMLGE AALTAADAER YRLAYAQAIT AIGKAATRGS IAANPGISIK LSALHPRYEV AQEARVMAEL VPVVRDLARA AARAGIALHI DAEEQDRLAL SLRVMAAVIA DPETAGWEGF GAVVQAYGKR AGAAIDALAA MARAAGRRIN IRLVKGAYWD AEMKRAQVEG HPGFPLFTSK TGTDVAYICL AAKLFGLNDC IYPQFATHNA HTVAAVLEMA AGRPFEFQRL HGMGARLHDI VLRETGGRCR IYAPVGAHRD LLAYLVRRLL ENGANSSFVN QIVNESVPPA EVAACPFAAL PTARAPRGLL APADLFGAGR VNAQGFDLSD PEVLARIEAA RDVTLPDAAP IVAGPVSGTL RPVRNPATGA VVAQVTEADA ATVALALDAA QVWSAPAATR AAVLCRAADL YEENFGPIFA ALAQEAGKTL GDAVSELREA VDFLRYYAAE GAADTRPPRG AVVAISPWNF PLAIFTGQVA AALMAGNAVL AKPAEQTPII AALAVRLLHQ AGVPETALQL LPGDGPTVGA ALTRDPRVAG VVFTGSTETA QIIARAMAAH LAPGTPLIAE TGGLNAMVVD STALPEQAVR DVVASAFRSA GQRCSALRCL YVQDDIAPHL IGMLKGAMEE LVSGDPARLS TDVGPVIDAE AKAGIETYLA ANKARILHRS TAPEGGHFVA PALLQVGGIA DLEREIFGPV LHLATFAAED LPAVIAAINA RGYGLTFGLH SRIDARVETV AETIRAGNIY VNRNQIGAVV GSQPFGGEGL SGTGPKAGGP LYLNRFYAPE PVVAVGGWTE AATPILPEAR ETQLDEIFLP GPTGELNRLT RHQRGPILCL GPGAEAASAQ AAAVVALGGQ AVQASGAVSP KALETLTPLA GVLWWGAAEM GRAYAQALAV RPGPLVPLIT AKPDLAHVAH ERHLCVDTTA AGGNAALLAG

Experimental information

• Beam: Instrument name: Advanced Light Source (ALS) 12.3.1 (SIBYLS); City: Berkeley, CA / 国: USA / Type of source: X-ray synchrotron / Wavelength: 0.1 Å / Dist. spec. to detc.: 1.5 mm
• Detector: Name: MAR 165 CCD

Scan

Title: Chimeric EcRHH-RcPutA / Measurement date: Oct 16, 2012 / Storage temperature: 4 °C / Cell temperature: 20 °C / Exposure time: 0.5 sec. / Number of frames: 2 / Unit: 1/A /

	Min	Max
Q	0.0112	0.3165

Distance distribution function P(R)

Sofotware P(R): GNOM 4.5a / Number of points: 503 /

	Min	Max
Q	0.01125	0.3165
P(R) point	1	503
R	0	183

Result

Type of curve: single_conc /

	Experimental	Porod
MW	251 kDa	-
Volume	-	308 nm3

	P(R)	P(R) error	Guinier	Guinier error
Forward scattering, I0	3033	5.5	3011.44	11
Radius of gyration, Rg	5.41 nm	0.008	5.2 nm	0.02

	Min	Max	Error
D	-	18.3	1
Guinier point	1	23	-