Kinetic, mutagenic, and structural homology analysis of l-serine dehydratase from Legionella pneumophila

Abstract

A structural database search has revealed that the same fold found in the allosteric substrate binding (ASB) domain of Mycobacterium tuberculosis D-3-phosphoglycerate dehydrogenase (PGDH) is found in l-serine dehydratase from Legionella pneumophila. The M. tuberculosis PGDH ASB domain functions in the control of catalytic activity. Bacterial l-serine dehydratases are 4Fe–4S proteins that convert l-serine to pyruvate and ammonia. Sequence homology reveals two types depending on whether their α and β domains are on the same (Type 2) or separate (Type 1) polypeptides. The α domains contain the catalytic iron–sulfur center while the β domains do not yet have a described function, but the structural homology with PGDH suggests a regulatory role. Type 1 β domains also contain additional sequence homologous to PGDH ACT domains. A continuous assay for l-serine dehydratase is used to demonstrate homotropic cooperativity, a broad pH range, and essential irreversibility. Product inhibition analysis reveals a Uni–Bi ordered mechanism with ammonia dissociating before pyruvate. l-Threonine is a poor substrate and l-cysteine and d-serine are competitive inhibitors with Ki values that differ by almost 10-fold from those reported for Escherichia colil-serine dehydratase. Mutagenesis identifies the three cysteine residues at the active site that anchor the iron–sulfur complex.