Structure-function relations in oxaloacetate decarboxylase complex. Fluorescence and infrared approaches to monitor oxomalonate and Na(+) binding effect.
BACKGROUND
Oxaloacetate decarboxylase (OAD) is a member of the Na(+) transport decarboxylase enzyme family found exclusively in anaerobic bacteria. OAD of Vibrio cholerae catalyses a key step in citrate fermentation, converting the chemical energy of the decarboxylation reaction into an electrochemical gradient of Na(+) ions across the membrane, which drives endergonic membrane reactions such as ATP synthesis, transport and motility. OAD is a membrane-bound enzyme composed of alpha, beta and gamma subunits. The alpha subunit contains the carboxyltransferase catalytic site.


RESULTS
In this report, spectroscopic techniques were used to probe oxomalonate (a competitive inhibitor of OAD with respect to oxaloacetate) and Na(+) effects on the enzyme tryptophan environment and on the secondary structure of the OAD complex, as well as the importance of each subunit in the catalytic mechanism. An intrinsic fluorescence approach, Red Edge Excitation Shift (REES), indicated that solvent molecule mobility in the vicinity of OAD tryptophans was more restricted in the presence of oxomalonate. It also demonstrated that, although the structure of OAD is sensitive to the presence of NaCl, oxomalonate was able to bind to the enzyme even in the absence of Na(+). REES changes due to oxomalonate binding were also observed with the alphagamma and alpha subunits. Infrared spectra showed that OAD, alphagamma and alpha subunits have a main component band centered between 1655 and 1650 cm(-1) characteristic of a high content of alpha helix structures. Addition of oxomalonate induced a shift of the amide-I band of OAD toward higher wavenumbers, interpreted as a slight decrease of beta sheet structures and a concomitant increase of alpha helix structures. Oxomalonate binding to alphagamma and alpha subunits also provoked secondary structure variations, but these effects were negligible compared to OAD complex.


CONCLUSIONS
Oxomalonate binding affects the tryptophan environment of the carboxyltransferase subunit, whereas Na(+) alters the tryptophan environment of the beta subunit, consistent with the function of these subunits within the enzyme complex. Formation of a complex between OAD and its substrates elicits structural changes in the alpha-helical as well as beta-strand secondary structure elements.
Granjon
Thierry
T
Institute of Microbiology, Eidgenössische Technische Hochschule, Zürich, Switzerland. thierry.granjon@univ-lyon1.fr
Maniti
Ofelia
O
Auchli
Yolanda
Y
Dahinden
Pius
P
Buchet
René
R
Marcillat
Olivier
O
Dimroth
Peter
P
D002262:Carboxy-Lyases / Q000737:chemistry / Q000378:metabolism*; D002384:Catalysis; D004563:Electrochemistry; D008314:Malonates / Q000378:metabolism*; D008958:Models, Molecular; D011485:Protein Binding; D017433:Protein Structure, Secondary; D012964:Sodium / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013055:Spectrophotometry, Infrared / Q000379:methods*; D013329:Structure-Activity Relationship
D008314:Malonates; D012964:Sodium; D002262:Carboxy-Lyases; C021916:oxaloacetate decarboxylase