VITAMINS

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This review describes the functional analysis based on the three dimensional structure of two unique enzymes, anti-tumor L-methinine γ-lyase and NAD dependent isopropylmalate dehydrogenase. L-Methionine γ-lyase (MGL, EC 4.4.1.11, also commonly referred to as methioninase) plays a central role in microbial methionine catabolism. MGL utilizes pyridoxal 5'-phosphate (PLP) as a cofactor and catalyzes manifold reactions such as α, γ-elimination and γ-replacement reactions of L-methionine and its derivatives, and also α, β-elimination and β-replacement reactions of S-substituted L-cysteines. MGL has an anti-tumor activity toward a broad variety of tumor cells. The application of its anti-tumor activity to creation of a novel type of anti-tumor reagent that has little side effect, is now in progress. We have cloned the MGL gene from Pseudomonas putida, constructed expression system, and established the rapid and large scale purification procedure for industrial use. We have determined two crystal structures of MGL using MAD(multiwavelength anomalous diffraction) and molecular replacement method. The overall structure of MGL is similar to those of other enzymes of transsulfuration via cystathionine, i.e. L-cystathionine β-lyase (CBL) and L-cystathionine γ-synthase (CGS). In spite of structural similarity, MGL doesn't utilize cystathionine as a substrate. This difference seem to be derived from the difference of amino acid residues which interact with substrate. We observed that Tyr114 and Cys116 in MGL, both residues have been considered to be very important for enzyme activity, are located at the active site of this enzyme and may play an important role on elimination reaction and substrate recognition. 3-Isopropylmalate dehydrogenase (IPMDH, EC 1.1.1.85), a key enzyme in leucine biosynthesis, catalyzes the NAD dependent oxidative decarboxylation of the substrate 3-isopropylmalate to 2-oxoisocaproate simultaneously with dehydrogenation. We constructed an overepression system of IPMDH from Thiobacillusferrooxidans in Escherichia coli. The crystal structure at 2.0Å resolution of IPMDH complexed with 3-isopropylmalate were determined by the molecular replacement method. The structure shows a fully closed conformation and substrate-binding site is quite similar to that of isocitrate dehydrogenase (ICDH) except for a region around the γ-isopropyl group. The γ group is recognized by a unique hydrophobic pocket, which includes Glu88, Leu9l and Leu92 from subunit 1 and Val 93' from subunit 2.

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