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Kinetic survey of evolutionary diverse isoforms of malate dehydrogenase
Author(s) -
Sampson Grant,
Chang Nathan,
Greene Erika,
Provost Joseph
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.633.27
Subject(s) - malate dehydrogenase , biochemistry , biology , citrate synthase , malic enzyme , enzyme , gene isoform , eubacterium , bacteria , dehydrogenase , gene , genetics
Malate dehydrogenase (MDH) catalyzes the redox formation of malate to oxaloacetate (OAA). Several isoforms exist. Mitochondrial MDH plays a well‐known role in the last step of the Krebs cycle while cytosolic MDH is involved in the NADH shuttle and other metabolic pathways. Plants, fungi, and protists express MDH in the glyoxisomes, where a variant of the Krebs cycle takes place. To better understand the unique enzymatic differences between evolutionarily distinct MDH isoforms, we have synthesized C‐terminal His tagged with a protease cleavage site and codon optimized 13 different isoforms of MDH for expression in E. coli . These include MDH from Chlorobaculum parvum , a green sulfur photosynthetic bacterium which uses sulfide, sulfur or thiosulfate as electronic donors; Streptomyces avermitilis , a soil bacterium with unique behaviors including insecticidal properties; saline tolerant Haloarcula marismortui , a Halophilic red Archaeon found in the Dead sea; Aquaspirillium arcticum bacterium which is cold tolerant living under the snow and ice of the pacific northwest; as well as all forms of yeast and human MDH. The purpose of this study is to evaluate structural and functional differences between each isoform of MDH, purified using affinity chromatography. Enzyme assays were performed to determine Km/Vmax of each isoform under varying OAA and NADH concentrations and to assess the inhibitory effects of citrate. Thermal stability assays were performed to assess resilience to heat, cold, and determine optimal storage conditions. A final cladogram was constructed to represent the evolutionary diversity between MDH isoforms. This work will help uncover the shifts in primary and structural evolutionary changes with functional kinetics of each MDH isoform and support use of these enzymes in the classroom. Support or Funding Information Funded in part by NSF IUSE DUE 1726932. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .