Functional Characterization of Key Enzymes involved in l-Glutamate Synthesis and Degradation in the Thermotolerant and Methylotrophic Bacterium Bacillus methanolicus

Abstract

ABSTRACTBacillus methanolicuswild-type strain MGA3 secretes 59 g/liter−1ofl-glutamate in fed-batch methanol cultivations at 50°C. We recently sequenced the MGA3 genome, and we here characterize key enzymes involved inl-glutamate synthesis and degradation. One glutamate dehydrogenase (GDH) that is encoded byyweBand two glutamate synthases (GOGATs) that are encoded by thegltABoperon and bygltA2were found, in contrast toBacillus subtilis, which has two different GDHs and only one GOGAT.B. methanolicushas a glutamine synthetase (GS) that is encoded byglnAand a 2-oxoglutarate dehydrogenase (OGDH) that is encoded by theodhABoperon. TheyweB,gltA,gltB, andgltA2gene products were purified and characterized biochemicallyin vitro. YweB has a lowKmvalue for ammonium (10 mM) and a highKmvalue forl-glutamate (250 mM), and theVmaxvalue is 7-fold higher forl-glutamate synthesis than for the degradation reaction. GltA and GltA2 displayed similarKmvalues (1 to 1.4 mM) andVmaxvalues (4 U/mg) for bothl-glutamate and 2-oxoglutarate as the substrates, and GltB had no effect on the catalytic activities of these enzymesin vitro. Complementation assays indicated that GltA and not GltA2 is dependent on GltB for GOGAT activityin vivo. To our knowledge, this is the first report describing the presence of two active GOGATs in a bacterium.In vivoexperiments indicated that OGDH activity and, to some degree, GOGAT activity play important roles in regulatingl-glutamate production in this organism.