Identification of key amino acid residues in the catalytic mechanism of diaminopropionate ammonialyase from Salmonella typhimurium

Abstract

Diaminopropionate ammonialyase (DAPAL), a fold-type II pyridoxal 5′-phosphate-dependent enzyme, catalyzes the a,b-elimination of diaminopropionate (DAP) to pyruvate and ammonia. DAPAL was able to utilize both D-and L-DAP as substrates with almost equal efficiency. Mutational analysis of functionally important residues such as Thr385, Asp125 and Asp194 was carried out to understand the mechanism by which the isomers are hydrolyzed. Further, the putative residues involved in the formation of disulfide bond Cys271 and Cys299 were also mutated. T385S, T385D sDA-PAL were as active with DL-DAP as substrate as sDAPAL, whereas the later exhibited a threefold increase in catalytic efficiency with D-Ser as substrate. Further analysis of these mutants suggested that DAPAL might follow an anti-E 2 mechanism of catalysis that does not involve the formation of a quinonoid intermediate. Of the two mutants of Asp125, D125E showed complete loss of activity with D-DAP as substrate, whereas the reaction with L-DAP was not affected significantly, demonstrating that Asp125 was essential for abstraction of protons from the D-isomer. By contrast, mutational analysis of Asp194 showed that the residue may not be directly involved in proton abstraction from L-DAP. sDAPAL does not form a disulfide bond in solution, although the position of Cys299 and Cys271 in the modeled structure of sDAPAL favored the formation of a disulfide bond. Further, unlike eDAPAL, sDAPAL could be activated by monovalent cations. Mutation of the cysteine residues showed that Cys271 may be involved in coordinating the monovalent cation, as observed in the case of other fold-type II enzymes.

L‐2,3‐diaminopropionate generates diverse metabolic stresses in Salmonella enterica

“…DpaL activity was measured by coupling pyruvate formation to NADH oxidation using lactate dehydrogenase (Sigma) and monitoring the decrease in absorbance at 340 nm as previously described (Kalyani et al, 2013). RidA was purified and its activity was confirmed previously (Ernst et al, 2014).…”

“…Expression of dpaL and protein production were specifically induced in S. enterica when exposed to Dap (Kalyani et al ., ). Furthermore, the narrow substrate range observed for DpaL in vitro and its favorable affinity for Dap ( K m = 0.1–0.3 mM) support a specific role for DpaL in degrading Dap to avoid metabolic stress (Nagasawa et al ., ; Khan et al ., ; Kalyani et al ., ).…”