Mechanistic aspects of the transamination reactions catalyzed by D-amino acid transaminase from Haliscomenobacter hydrossis |
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Institution: | 1. A.N.Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Leninsky Ave. 33, bld. 2, 119071 Moscow, Russian Federation;2. Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Kosygin st., 4, 119334 Moscow, Russian Federation |
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Abstract: | Pyridoxal-5′-phosphate-(PLP-) dependent D-amino acid transaminases (DAATs) catalyze stereoselective reversible transfer of the amino group between D-amino acids and keto acids. In vivo DAATs are commonly known to synthesize D-glutamate for cell wall peptidoglycans. Today DAATs meet increasing attention for application in the synthesis of D-amino acids, whereas little is known about the mechanism of substrate recognition and catalytic steps of the D-amino acids conversion by DAATs. In this work, the pre-steady-state kinetics of the half-reactions of DAAT from Haliscomenobacter hydrossis with D-glutamate, D-alanine, D-leucine, and D-phenylalanine was examined at two wavelengths, 416 and 330 nm, using a stopped-flow technique. Monophasic kinetics was observed with specific substrates D-glutamate and D-alanine, whereas half-reactions with D-leucine and D-phenylalanine exhibited biphasic kinetics. All half-reactions proceeded until the complete conversion of PLP due to the release of the pyridoxamine-5′-phosphate form of cofactor from the holoenzyme . Comparison of kinetic parameters of half-reactions and the overall transamination reactions for D-leucine, D-phenylalanine revealed the increase in the rates of deamination of these substrates in the overall reaction with α-ketoglutarate. In the overall transamination reaction, the catalytic turnover rates for D-leucine and D-phenylalanine increased by 260 and 60 times, correspondingly, comparing with the slowest step rate constants in the half-reactions. We suggested the activating effect by a specific substrate α-ketoglutarate in the overall transamination reaction. The study of half-reactions helped to quantify the specificity of DAAT from H. hydrossis for D-amino acids with different properties. The results obtained are the first detailed analysis of half-reactions catalyzed by DAAT. |
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Keywords: | PLP"} {"#name":"keyword" "$":{"id":"pc_Q0TQG4zjdy"} "$$":[{"#name":"text" "_":"pyridoxal-5′-phosphate PMP"} {"#name":"keyword" "$":{"id":"pc_nqAr7viWUc"} "$$":[{"#name":"text" "_":"pyridoxamine-5′-phosphate DAAT"} {"#name":"keyword" "$":{"id":"pc_OcwkzvY0Sz"} "$$":[{"#name":"text" "_":"D-amino acid transaminase Halhy"} {"#name":"keyword" "$":{"id":"pc_aP3wEySTMJ"} "$$":[{"#name":"text" "$$":[{"#name":"__text__" "_":"D-amino acid transaminase from "} {"#name":"italic" "_":"Haliscomenobacter hydrossis TA"} {"#name":"keyword" "$":{"id":"pc_gGUvQZoVkB"} "$$":[{"#name":"text" "_":"transaminase |
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