FIG. 4.

Pirin_Sm inhibits PDH E1 and PDH enzyme complex activities. (A) Effect of pirin_Sm on PDH E1_Sm specific activity. E. coli BL21(DE3) was used as the host for protein synthesis. (B) Effect of pirin_Sm gene overexpression (0.02% arabinose induction) from pSC15 on PDH E1_Sm specific activity in S. marcescens CH-1. (C) Effect of pirin_Sm gene deletion on PDH E1_Sm specific activity in S. marcescens CH-1. (D) Effect of pirin_Sm gene overexpression (0.02% arabinose induction) from pSC15 on PDH E1_Sm specific activity in S. marcescens PC103. (E) Purification of S. marcescens PDH E1, E2, and E3 subunits in E. coli DH5α cells. Lane 1, GST-E1 subunit (pSC12); lane 2, GST-E2 subunit (pSC19); lane 3, GST-E3 subunit (pSC20); lane 4, GST-pirin (pSC10); lane 5, GST-NlpB_Sm (pSC11); 6, GST protein (pGEX vector). (F) Effect of purified pirin_Sm, NlpB_Sm, and GST (15 μg each) on specific activity of purified PDH E1 subunit (15 μg). (G) PDH enzyme complex activity in S. marcescens CH-1 and PC103. (H) Effect of pirin_Sm gene overexpression (0.02% arabinose induction) from pSC15 on specific PDH enzyme complex activity in S. marcescens PC103. (I) Effect of pirin_Sm on reconstituted PDH enzyme complex at an E1/E2/E3 molar ratio of 2:2:1. The DCPIP assay (12) was used to measure PDH E1 activity; the rate of NADH formation was measured spectrophotometrically at a wavelength of 340 nm (5) to measure PDH enzyme complex activity. Black and white bars indicate reactions with or without pyruvate added as the substrate. PBS (pH 7.4) was used as the negative control for all assays. CH-1, S. marcescens CH-1; PC103, a pirin_Sm gene insertion-deletion mutant strain derived from S. marcescens CH-1; pBAD18, pBAD18-Kan control vector; pSC15, pBAD18-Kan::pirin_Sm gene; NlpB_Sm, a membrane lipoprotein identified in S. marcescens CH-1 (28). a, change in OD₆₀₀ per minute per milligram of protein. b, change in OD₃₄₀ per minute per milligram of protein. Values are means and standard deviations from three independent experiments. *, P < 0.05.