Methemoglobin reduction mediated by d-amino acid dehydrogenase in Propsilocerus akamusi (Tokunaga) larvae

• d-Amino acid dehydrogenase (DAD) was found in a microaerobic multicellular organism.
• DAD mediated a novel methemoglobin reduction in Propsilocerus akamusi larvae.
• The reduction system is composed of d-alanine, DAD, and cytochrome b5.

A methemoglobin (metHb) reduction system is required for aerobic respiration. In humans, Fe(III)-heme-bearing metHb (the oxidized form of hemoglobin), which cannot bind oxygen, is converted to Fe(II)-heme-bearing oxyhemoglobin (oxyHb, the reduced form), which can bind oxygen, in a system comprising NADH, NADH-cytochrome b5 reductase, and cytochrome b5. However, the mechanism of metHb reduction in organisms that inhabit oxygen-deficient environments is unknown. In the coelomic fluid of the larvae of Propsilocerus akamusi, which inhabit a microaerobic environment, we found that metHb was reduced by d-alanine. We purified an FAD-containing enzyme, d-amino acid dehydrogenase (DAD), and component V hemoglobin from the larvae. Using the purified components and spectrophotometric analyses, we showed a novel function of DAD: DAD-mediation of P. akamusi component V metHb reduction with using d-alanine as an electron donor. P. akamusi larvae possess this d-alanine–DAD metHb reduction system in addition to a previously discovered NADH–NADH-cytochrome b5 reductase system. This is the first report of the presence of DAD in a multicellular organism. The molecular mass of DAD was estimated to be 45 kDa. The optimal pH and temperature of the enzyme were 7.4 and 20 °C, respectively, and the optimal substrate was d-alanine. The enzyme activity was inhibited by benzoate and sulfhydryl-binding reagents.

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