Abstract <p>Membrane-bound pyrroloquinoline quinone (PQQ)–dependent quinoproteins are key components of the periplasmic oxidative metabolism of acetic acid bacteria, yet several predicted enzymes remain experimentally uncharacterized. Here, we identified and characterized Pqq5 from <i>Gluconobacter japonicus</i> NBRC 3271 as a membrane-bound PQQ-dependent D-lactate dehydrogenase (mLDH). Overexpression of mLDH in a strain lacking six major membrane bound dehydrogenases markedly increased D-lactate dehydrogenase activity in membrane fractions. The enzyme showed a clear preference for D-lactate over L-lactate, exhibited maximal activity at pH 6.0, and retained substantial activity at pH 4.0–6.0. Kinetic analysis using membrane fractions yielded an apparent <i>K</i>_M of 1.08 mM for D-lactate. Membrane fractions containing mLDH showed ubiquinone-1 reductase activity and D-lactate-dependent oxygen consumption, suggesting that the enzyme is involved in quinone-linked D-lactate oxidation. D-lactate dehydrogenase activity was decreased by EDTA treatment, and restored by supplementation with PQQ and CaCl₂, supporting the assignment of mLDH as a quinoprotein. In addition, pyruvate was produced from D-lactate in an mLDH-dependent manner. In an engineered background lacking known D-lactate-oxidizing enzymes, overexpression of mLDH helped growth on D-lactate medium. Together, these results support the identification of Pqq5 as a membrane-bound PQQ-dependent D-lactate dehydrogenase in <i>G. japonicus</i> and suggest that this enzyme contributes to quinone-linked D-lactate utilization.</p> Key points <p>• Pqq5<i> is a membrane-bound PQQ-dependent D-lactate dehydrogenase, mLDH</i></p> <p>• <i>mLDH catalyzes D-lactate oxidation and ubiquinone reduction in membrane fractions</i></p> <p>• <i>mLDH helps growth on D-lactate in an engineered G. japonicus background</i></p>