Crystal Structures and Molecular Dynamics Simulations of Thermophilic Malate Dehydrogenase Reveal Critical Loop Motion for Co-Substrate Binding

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Title:	Crystal Structures and Molecular Dynamics Simulations of Thermophilic Malate Dehydrogenase Reveal Critical Loop Motion for Co-Substrate Binding
Authors:	Hung, CH (Hung, Chih-Hung) Hwang, TS (Hwang, Tzann-Shun) Chang, YY (Chang, Yu-Yung) Luo, HR (Luo, Huei-Ru) Wu, SP (Wu, Szu-Pei) Hsu, CH (Hsu, Chun-Hua)
Contributors:	Grad Inst Biotechnol
Keywords:	THERMUS-THERMOPHILUS NUCLEOTIDE-SEQUENCE OVEREXPRESSION PURIFICATION GENE EVOLUTION CLONING ENZYME FLAVUS
Date:	2013-12-26
Issue Date:	2014-02-21 11:07:21 (UTC+8)
Abstract:	Malate dehydrogenase (MDH) catalyzes the conversion of oxaloacetate and malate by using the NAD/NADH coenzyme system. The system is used as a conjugate for enzyme immunoassays of a wide variety of compounds, such as illegal drugs, drugs used in therapeutic applications and hormones. We elucidated the biochemical and structural features of MDH from Thermus thermophilus (TtMDH) for use in various biotechnological applications. The biochemical characterization of recombinant TtMDH revealed greatly increased activity above 60 degrees C and specific activity of about 2,600 U/mg with optimal temperature of 90 degrees C. Analysis of crystal structures of apo and NAD-bound forms of TtMDH revealed a slight movement of the binding loop and few structural elements around the co-substrate binding packet in the presence of NAD. The overall structures did not change much and retained all related positions, which agrees with the CD analyses. Further molecular dynamics (MD) simulation at higher temperatures were used to reconstruct structures from the crystal structure of TtMDH. Interestingly, at the simulated structure of 353 K, a large change occurred around the active site such that with increasing temperature, a mobile loop was closed to co-substrate binding region. From biochemical characterization, structural comparison and MD simulations, the thermal-induced conformational change of the co-substrate binding loop of TtMDH may contribute to the essential movement of the enzyme for admitting NAD and may benefit the enzyme's activity.
Relation:	PLOS ONE Volume: 8 Issue: 12 Article Number: e83091
Appears in Collections:	[Graduate Institute of Biotechnology ] journal articles

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