부산대학교 도서관

R5499 --> 795.1 --> Burkholderia cenocepaciais an opportunistic pathogen posing risk among individuals with cystic fibrosis or who are immune compromised. The crystal structure of a putative hydroxybutyrate dehydrogenase (HBDH), based on sequence analysis, from Burkholderia cenocepaciahas been solved by the Seattle Structural Genomics Center for Infectious Disease and deposited in the Protein Database (PDB ID 4TRR). We sought to determine if the protein is an HBDH and to determine the kinetic parameters of the enzyme. HBDHs catalyze the reversible conversion of acetoacetate to hydroxybutyrate utilizing the cofactor NAD(H). The SSGCID kindly provided us the construct encoding a His‐tagged BcHBDH. The protein was overexpressed in E. coli, purified to approximate homogeneity using nickel affinity chromatography, and activity assayed spectrophotometrically. The putative BcHBDH is confirmed to have HBDH activity and the kinetic parameters for the enzyme were determined in both the direction of acetoacetate and hydroxybutyrate formation. BcHBDH has a catalytic efficiency of 2.85x104 mM‐1 sec‐1 and 1.21x105mM‐1 sec‐1 in the direction of hydroxybutyrate formation for acetoacetate and NADH, respectively. In the direction of acetoacetate formation, the enzyme has a catalytic efficiency of 4.1x103 mM‐1 sec‐1 and 2.86x104 mM‐1 sec‐1 for hydroxybutyrate and NAD+, respectively. By comparing both the structure and kinetic parameters to known kinetically characterized and crystallized HBDHs, we conclude the protein from Burkholderia cenocepacia is an HBDH. [ABSTRACT FROM AUTHOR]