The molecular basis of an osmotically reparable mutant of Neurospora crassa producing unstable glutamate dehydrogenase

Mutant 14 in the am gene of Neurospora crassa, coding for NADP-specific glutamate dehydrogenase (E.C.1.4.1.4), has been previously shown to complement positively with several other am mutants and negatively with the wild type, but to produce no detectable glutamate dehydrogenase protein under normal conditions of growth. We show in this paper that am14 is partially repaired by 1.0 to 1.5 m-glycerol, glucose, mannitol or sorbitol, or 0.5 to 0.75 m-KCl in the growth medium so as to produce highly unstable glutamate dehydrogenase. Two kinds of pseudo-wild revertants from am14 produce partially stabilized glutamate dehydrogenase varieties on normal medium but their enzyme formation is still increased by glycerol in the growth medium. The formation of the enzyme in am14 and in both types of revertant is highly temperature-sensitive. Amino acid sequence analysis of the enzyme varieties formed by the two revertant types shows the replacement Leu20 → His in one case (R5) and Leu20 → Tyr in the other (R1). It is deduced by coding considerations that the primary replacement in am14 itself must be Leu20 → His, and that R5 has a partially compensating replacement at a second unknown residue, while R1 has a second change in the same residue, Leu20 → His → Tyr. The properties of the am14 enzyme are attributed to unstable quaternary structure due to the repacement of a hydrophobic side chain (leucine) by a polar one (histidine) at or near a point of contact between monomers in the enzyme hexamer. It is speculated that the relative stabilization brought about in growth media of high osmolality is due to the reduction on the effective concentration of water molecules surrounding the protein, with a consequent reduction of the disruptive effect of the polar side chain.