The C-terminal regulatory domain is required for catalysis by Neisseria meningitidis alpha-isopropylmalate synthase.

ABSTRACT

alpha-Isopropylmalate synthase (alpha-IPMS) catalyses the first committed step in leucine biosynthesis in many pathogenic bacteria, including Neisseria meningitidis. This enzyme (NmeIPMS) has been purified, characterised, and compared to alpha-IPMS proteins from other bacteria. NmeIPMS is a homodimer which catalyses the condensation of alpha-ketoisovalerate (alpha-KIV) and acetyl coenzyme A (AcCoA), and is inhibited by leucine. NmeIPMS can use alternate alpha-ketoacids as substrates and, in contrast to alpha-IPMS from other sources, is activated by a range of metal ions including Cd(2+) and Zn(2+) that have previously been reported as inhibitory, since they suppress the dithiodipyridone assay system rather than the enzyme itself. Previous studies indicate that alpha-IPMS is a TIM barrel enzyme with an allosteric leucine-binding domain. To assess the importance of this domain, a truncated form of NmeIPMS was generated and characterised. Loss of the regulatory domain resulted in a loss of the ability to catalyse the aldol reaction, although the enzyme was still able to slowly hydrolyse AcCoA independently of alpha-KIV at a rate similar to that of the WT enzyme. This implies that the regulatory domain is not only required for control of enzymatic activity but may assist in the positioning of key residues in the catalytic TIM barrel. The importance of this domain to catalytic function may offer new strategies for inhibitor design.