A Functional Genomics Approach To Analyze Two Methionine-[Gamma]-Lyase Orthologs In "Ferroplasma Acidarmanus" Using In Vitro Enzyme Assays

Abstract

Functional genomics predicts that ferl genome has two candidate genes for the production of two methionine-y-lyase (MGL) orthologs, which are thought to be pivotal for the production of methanethiol. These orthologs show a high level of protein sequence identity ( ~ 3 5%) and similarity (~60%) when compared to known MGLs. In addition, ClustalW2 alignment of ferl MGL orthologs and other known MG Ls showed a high level of similarity in amino acid residues, which are involved for the stabilization of 3D structure of MGL and in forming active site for the substrates. To confirm the predictive functions of these two orthologs, enzymatic assays using ferl cell lysate as well as E. coli BL21 (DE3) cell lysates having these orthologs were carried out. The break down products of L-methionine by MGL includes methanethiol and and/or a-keto butyrate. In the first assay, DTNB (5, 5'-dithiobis-(2-nitrobenzoic acid) was used to detect compounds containing thiol (-SH) group, such as methanethiol. The second assay uses MBTH (3-methyl-2-benzothiazolinone) to detect the presence of alpha-keto acids. In DTNB assays, ferl lysate produced thiol-containing compounds in a time-temperature dependent reaction (N=4). The lack of activity in heat-treated lysate controls showed that the reaction was enzymatic. Moreover, these activities were dependent on both pyridoxal phosphate (PLP), a coenzyme of previously reported MG Ls from other sources, and on the addition of L-methionine as substrate. Using the ferl cell lysates, the highest specific activity (0.14 μmol/mg/min, N=5) was detected at pH 4.0 in MBTH assay. As with the L-methionine, substrate specificities of ferl lysate were studied. D-methionine, L-cysteine, L-cystathionine and DL-homocysteine were degraded to 97.5%, 130.2%, 88.9%, 152.5% respectively of the level of activity on L-Met. In order to study individually, these MGL candidates have been cloned in pET21 b vectors and transferred in E. coli BL21 (DE3). The cell lysates of E. coli expressing these orthologs were tested for MGL activities. For MBTH and DTNB assays, the highest specific activity of MGLl, 0.19 μmol/mg/min (N=4 and N=2, respectively) was detected at pH 4 and at pH 5, respectively.

Date of Award	2011
Original language	American English
Awarding Institution	Eastern Illinois University
Supervisor	Billy Hung (Supervisor)