Location and characteristics of enzymes involved in the breakdown of polygalacturonic acid by Bacteroides thetaiotaomicron

Abstract

When Bacteroides thetaiotaomicron is grown in medium which contains polygalacturonic acid (PGA) as the sole carbon source, two different polygalacturonases are produced: a PGA lyase (EC 4.2.2.2) and a PGA hydrolase (EC 3.2.1.15). Both enzymes are cell associated. The PGA hydrolase appears to be an inner membrane protein. The PGA lyase is a soluble protein that associates with membranes under certain conditions. The PGA lyase was purified to apparent homogeneity. It has a molecular weight (from sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of 74,000, a pH optimum of 8.7, a pI of 7.5, and a K(m) for PGA of 40 to 70 μg/ml. It requires calcium for maximal activity. The main product of this enzyme appears to be a disaccharide that contains a Δ4,5-unsaturated galacturonic acid residue. The PGA hydrolase can be solubilized from membranes with 2% Triton X-100 and has been partially purified. It has a pH optimum of 5.4 to 5.5, a pI of 4.7 to 4.9, and a K(m) for PGA of 350 to 400 μg/ml. The main product of this enzyme appears to be galacturonic acid. The specific activities of both PGA hydrolase and PGA lyase increase at the same rate when bacteria are exposed to PGA. The two enzymes therefore appear to be similarly regulated.