Abstract
Proteus vulgaris L-amino acid deaminase (pvLAAD) belongs to a class of bacterial membrane-bound LAADs mainly express in genus Proteus, Providencia and Morganella. These LAADs employ a non-cleavable N-terminal twin-arginine translocation (Tat) peptide to transport across membrane and bind to bacterial surface. Recent studies revealed that a hydrophobic insertion sequence (INS) in these LAADs also interacts with bacterial membrane. However, the functional significance of INS-membrane interaction is not clear. In this study, we made site-directed mutagenesis on the surface-exposed hydrophobic residues of pvLAAD INS, and we found that these mutations impaired the INS-membrane interaction but did not affect pvLAAD activity in the solution. We further found that when cell membrane is present, the catalytic activity can be increased by 8~10 folds for wild-type but not INS-mutated pvLAAD, indicating that the INS-membrane interaction is necessary for increasing activity of pvLAAD. Molecular dynamic (MD) simulations suggested that INS is flexible in the solution, and its conformational dynamics could lead to substrate channel distortion. Circular dichroism (CD) spectroscopy experiments indicated that bacterial membrane was able to maintain the conformation of INS. Our study suggests the function of the membrane binding of INS is to stabilize pvLAAD structure and increase its catalytic activity.
Highlights
L-amino acid oxidases/deaminases (LAAOs/LAADs, EC 1.4.3.2) are flavin-containing enzymes that catalyze the oxidative deamination of L-amino acids to corresponding α-keto acids with strict stereospecificity[1]
To figure out whether it is the sole mechanism for the activity enhancement by membrane, we detected the catalytic activity of Proteus vulgaris L-amino acid deaminase (pvLAAD) associated with the membrane whose electron transfer chain was blocked or not
When 10 mM of sodium azide, an inhibitor of cytochrome oxidase of the electron transfer chain[27], was added, the activity of pvLAAD with membrane was only slightly reduced (6382 ± 236 μM of phenylpyruvic acid (PPA) in 60 min), and still remained 7.8-fold higher than that of pvLAAD without membrane (Fig. 1a). This suggested that a mechanism other than the electron transfer chain-coupling is involved in membrane-caused activity enhancement of pvLAAD
Summary
L-amino acid oxidases/deaminases (LAAOs/LAADs, EC 1.4.3.2) are flavin-containing enzymes that catalyze the oxidative deamination of L-amino acids to corresponding α-keto acids with strict stereospecificity[1]. While most LAAOs/LAADs are secreted or cytosolic enzymes[8], bacteria from genus Proteus, Providencia and Morganella express a class of membrane-bound LAADs9 Studies revealed that these membrane-bound LAADs could produce α-keto acids as siderophores to capture irons from the environment. Another study showed that the plasmid containing the LAAD gene was unable to restore the iron-limiting survival of a siderophore negative E. coli strain[13], which put the physiological role of LAADs in debate These LAADs are gaining increasing interests for their potential applications on producing various α-keto acids in eco-friendly manners. The crystal structures of bacterial membrane-bound LAADs from Proteus myxofaciens (pmaLAAD) and Proteus vulgaris (pvLAAD) have been reported[21,25] This opens a door to understand the catalytic mechanism of this class of enzymes and invent the improved biocatalysts[26].
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