Exploration of Malate Dehydrogenase Structure and Functionality

Alex Hannappel Author

04/05/2021 Added

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Throughout your body and the rest of life, malate dehydrogenase is not only active but essential to life. Despite its prevalence and importance, the mechanisms behind this enzyme are not well. To end that lack of understanding, my research creates structural mutants to see how the changes affect the enzyme's function.

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[00:00:01.000]Hi, I'm Alexander Hannappel
[00:00:02.500]I senior biochemistry researcher in
[00:00:04.100]Dr.
[00:00:04.400]Jing Zhang's lab
[00:00:05.800]my research focuses on the
[00:00:06.990]exploration of malate
[00:00:07.700]dehydrogenase's structure and
[00:00:09.400]functionality.
[00:00:10.800]MDH for shorthand, is present in
[00:00:12.400]all organisms and is critical to
[00:00:14.200]survival
[00:00:15.300]Though
[00:00:15.500]MDH comes in a diversity of forms,
[00:00:17.800]they all retain a consistent
[00:00:18.410]structure
[00:00:19.300]that's still a mystery to science
[00:00:21.200]to change that, experiments like
[00:00:22.300]this one test how changes to an
[00:00:24.100]enzyme
[00:00:25.100]affect its reaction
[00:00:27.400]MDH operates in every corner of
[00:00:29.000]your body as as an essential
[00:00:31.000]metabolic enzyme
[00:00:31.900]In the Krebs cycle,
[00:00:32.700]It is partially responsible for
[00:00:34.200]collecting chemical energy.
[00:00:36.100]The energy is locked away in the
[00:00:37.400]form of electrons that must be
[00:00:38.700]collected in steps
[00:00:40.000]so as not to release a sudden
[00:00:40.910]burst of heat
[00:00:42.400]to do this.
[00:00:43.000]MDH harvest these electrons and
[00:00:44.700]passes them off to the compound NAD+
[00:00:47.600]the animation shows the reaction
[00:00:48.700]catalyzed by MDH
[00:00:51.200]in it NAD+ on the left gains an
[00:00:53.100]electron from malate: forming NADH
[00:00:55.680]and oxaloacetate. The Oxaloacetate
[00:00:57.900]continues through the (Krebs)
[00:00:58.690]cycle while NADH is destined to go
[00:01:01.500]towards the production of ATP, the
[00:01:03.500]body's energy currency that pays
[00:01:05.200]for cellular processes
[00:01:07.500]this collection of electrons, is
[00:01:09.200]ultimately what sustains life
[00:01:11.300]since the reaction is reversible
[00:01:12.200]MDH can play a critical role in
[00:01:14.700]the malate-aspartate shuttle where
[00:01:16.600]the conversion between malate and
[00:01:17.900]oxaloacetate shuttles high energy
[00:01:20.100]electrons into the mitochondria
[00:01:22.100]from the cytosol.
[00:01:24.600]MDH also has a darker side. Because
[00:01:26.800]of its universality, MDB is
[00:01:28.400]frequently implicated in the most
[00:01:29.600]devastating conditions
[00:01:31.400]Cancer cells heavily utilized
[00:01:33.000]MDH, especially while metastasizing.
[00:01:35.600]Chemotherapies targeting MDH would
[00:01:37.500]hamper cancer cell's ATP
[00:01:38.800]production and other critical
[00:01:40.200]cellular processes
[00:01:42.100]Increase flux through the
[00:01:42.900]Malate-aspartate shuttle is
[00:01:44.400]neurotoxic. On top of causing this
[00:01:46.400]damage,
[00:01:47.000]MDH is also it's victim Brain
[00:01:49.000]samples from Alzheimer's patients
[00:01:50.200]showed
[00:01:51.100]MDH structurally altered making it
[00:01:53.300]more likely to form
[00:01:54.600]brain, damaging plaques seen in
[00:01:56.100]Alzheimer's
[00:01:58.000]A highly conserved area of MDH is
[00:01:59.700]the mobile loop region.
[00:02:00.900]I gate-like feature that opens for
[00:02:02.200]reactants to enter and closes
[00:02:03.700]once they're bound. This spot is
[00:02:05.000]thought to function as a barrier
[00:02:05.610]protecting the active site where
[00:02:08.360]the reaction occurs. The roles of
[00:02:09.000]each amino acid here are not well
[00:02:10.300]understood.
[00:02:11.400]There's an old biochemistry adage
[00:02:12.400]that structure determines function.
[00:02:14.600]So knowing the chemical environment
[00:02:16.000]around an amino acid is critical to
[00:02:18.000]understanding the enzyme itself.
[00:02:19.900]Unfortunately, there's no way to
[00:02:20.900]directly observe the reaction at
[00:02:23.200]the molecular level. Instead,
[00:02:25.200]biochemists mutate a proteins
[00:02:26.510]amino acid sequence
[00:02:28.700]and then measure the resulting
[00:02:29.500]effect. Before that can be
[00:02:31.600]accomplished, an evaluation of the
[00:02:34.210]enzymes evolution and 3D model is
[00:02:36.700]used to locate residues of
[00:02:38.200]interest. The initial narrowing of
[00:02:39.700]this search, is done by comparing
[00:02:40.600]the amino acid sequences
[00:02:43.200]of different MDH versions and
[00:02:44.400]related enzymes
[00:02:46.600]Existing throughout life is
[00:02:47.500]indicative of a critical residue.
[00:02:49.500]Because of evolution's selection of
[00:02:50.900]the loop region across
[00:02:52.400]life the residues here must be
[00:02:54.100]important to the enzymes operation.
[00:02:56.600]Amino acid residue number 137 is
[00:02:59.500]conserved across related enzymes
[00:03:00.800]also containing the loop region
[00:03:03.500]Structural evidence indicates
[00:03:05.100]this residue may act as a lock and
[00:03:06.800]key by stabilizing the active site
[00:03:08.600]after the reactants have bound
[00:03:10.900]3D models help visualize an amino
[00:03:12.210]acid's position and chemical
[00:03:14.290]environment within an enzyme This
[00:03:15.710]enables researchers to refine
[00:03:17.300]their mutation selection.
[00:03:19.600]The left model shows the natural
[00:03:20.900]amino acid at this position:
[00:03:23.200]asparagine.
[00:03:24.500]The blue lines represent strong
[00:03:25.500]interactions called hydrogen-bonds.
[00:03:28.000]It suggests asparagine may be
[00:03:29.500]involved in the stabilization of
[00:03:31.100]the active site nearby causing the
[00:03:33.190]lock and key effect for the loop.
[00:03:35.000]To test this hypothesis, residue
[00:03:36.200]137 will be mutated to different
[00:03:38.200]amino acids to measure the effect
[00:03:39.810]on the reaction.
[00:03:41.300]The 20 amino acids have incredible
[00:03:42.600]overlap and also stark differences
[00:03:46.000]Mutating residue reveals the
[00:03:47.300]significance of different
[00:03:48.800]structural elements. The choice
[00:03:51.400]of amino acid here was made based
[00:03:53.010]on similarities to focus on very
[00:03:55.200]small elements.
[00:03:57.100]Glutamine and aspartic acid are
[00:03:58.300]close amino acid cousins to
[00:03:59.800]asparagine
[00:04:00.900]Glutamine, in the middle is one
[00:04:02.500]carbon longer than Asparagine.
[00:04:04.300]This mutation would suggest the
[00:04:05.700]role that size may play at the
[00:04:07.100]position
[00:04:08.700]here.
[00:04:09.000]It also has no stabilizing hydrogen
[00:04:11.100]bonds and may not even fit in the
[00:04:13.100]site based on the
[00:04:15.000]yellow line displayed.
[00:04:16.800]Aspartic acid on the right is
[00:04:18.100]similar in length to asparagine,
[00:04:19.490]but with different functional
[00:04:20.100]groups, which changes the chemical
[00:04:22.100]environment of the site
[00:04:23.800]It's model shows
[00:04:24.600]it fitting in the site but
[00:04:25.600]experiences.
[00:04:26.400]No stabilizing
[00:04:27.000]bonds.
[00:04:28.100]After reaction tests are conducted
[00:04:29.110]this information will help in
[00:04:30.900]hypothesizing the reason for any
[00:04:32.500]observed change
[00:04:34.400]Understanding how one position
[00:04:36.400]like this works provides a very
[00:04:37.800]small but critical snapshot of an
[00:04:39.700]enzyme
[00:04:40.600]methodically mutating across the
[00:04:42.000]whole enzyme then compiles those
[00:04:43.700]into a larger picture of how the
[00:04:45.300]enzyme works.
[00:04:47.200]watermelon glyoxysomal
[00:04:49.200]malate dehydrogenase or wgMDH is a
[00:04:52.300]close cousin of human forms that
[00:04:53.800]doesn't require additional
[00:04:54.600]assistance in becoming operational,
[00:04:56.900]making it ideal for this experiment.
[00:04:58.900]Every residue is represented in DNA
[00:05:01.100]as 3 specific three letter codon
[00:05:02.600]which, when altered, produces a
[00:05:04.100]mutant enzyme. To do this, a
[00:05:06.590]circular piece of MDH DNA is used
[00:05:09.200]as a template
[00:05:10.900]for the Polymerase Chain Reaction
[00:05:13.300]During the annealing step short
[00:05:14.500]pieces of mRNA containing the
[00:05:16.300]mutation
[00:05:17.000]stick to the clone
[00:05:18.400]The pair is replicated, producing
[00:05:20.190]copies of the mutant during
[00:05:21.300]extension
[00:05:22.500]The enzyme DPN1 then degrades the
[00:05:24.400]original DNA leaving only the mutant
[00:05:27.700]The mutated Gene is produced en
[00:05:28.910]masse
[00:05:29.800]once introduced to DNA expressing E.
[00:05:33.100]coli. After the cells are
[00:05:33.800]centrifuged away and the DNA
[00:05:35.100]purified E.coli engineered for
[00:05:37.300]protein expression are then used
[00:05:38.700]to express the gene
[00:05:41.000]Once given time to multiply and
[00:05:42.300]make the mutant enzyme,
[00:05:43.600]these can then get spun down to
[00:05:44.900]collect a cellular mass called a
[00:05:46.700]pellet containing the mutant enzyme
[00:05:49.400]The pellet then goes through a
[00:05:50.910]gauntlet of chemicals and sound
[00:05:52.300]ripping apart cells and DNA while
[00:05:54.100]proteins remain unaffected.
[00:05:55.900]Another round of intense
[00:05:57.210]centrifugation separates the solid
[00:05:59.400]cellular material from the crude.
[00:06:00.800]This liquid is then put through a
[00:06:02.300]specialized funnel intended to
[00:06:04.000]gradually isolate the mutant.
[00:06:05.300]MDH with a series of histidine
[00:06:07.500]amino acids or "His-tag" on the
[00:06:10.380]mutant enzyme makes it stick to
[00:06:11.800]the nickel-ions lining the column.
[00:06:13.700]Undesired compounds flow through
[00:06:15.700]the funnel while the enzyme
[00:06:17.300]remains. The enzyme is then
[00:06:19.100]displaced
[00:06:19.500]from the nickel using solution
[00:06:21.500]with immidazole
[00:06:22.880]the result is a mutant containing
[00:06:24.000]liquid called the eluent
[00:06:26.300]the sample goes through SDS page to
[00:06:28.300]verify the presence of MDH
[00:06:30.400]an unmutated or wildtype(wt)
[00:06:31.500]version of the enzyme is also
[00:06:33.200]purified for comparison during
[00:06:34.900]kinetics
[00:06:37.100]enzyme kinetics are the measurable
[00:06:38.010]qualities of reaction
[00:06:39.700]this test use the conversion of
[00:06:40.700]oxaloacetate to malate consuming
[00:06:42.100]NADH
[00:06:43.900]because of the difference between
[00:06:44.600]NAD+ and NADH. The reaction is
[00:06:47.900]measured using
[00:06:48.900]a 340 nanometer light source
[00:06:51.400]passed through the reaction vessel.
[00:06:53.600]The result is a graph of absorbance
[00:06:55.900]versus time that provides data that
[00:06:57.500]can graph the specific activity
[00:06:58.900]velocity and efficiency of an
[00:07:01.000]enzyme. Measurements for the
[00:07:02.600]mutant strains are then compared
[00:07:03.800]to the unmutated form.
[00:07:07.100]The experiment is ongoing but data
[00:07:08.600]on aspartic acid has been
[00:07:09.800]collected these results indicate a
[00:07:11.900]dramatic reduction in enzymatic
[00:07:13.790]activity, maximum reaction velocity,
[00:07:16.500]and efficiency.
[00:07:17.800]Overall, this indicates reduced
[00:07:19.000]functionality supporting the
[00:07:20.700]hypothesis that asparagine 137 is
[00:07:23.000]structurally important
[00:07:24.600]since models showed zero
[00:07:25.900]stabilizing interactions with
[00:07:27.700]aspartic acid at this position.
[00:07:30.200]It is suggested that asparagine
[00:07:31.200]bonds here are essential to the
[00:07:33.510]reaction.
[00:07:34.600]However, more aspartic acid trials
[00:07:35.900]and evaluation of glutamine
[00:07:37.500]mutants is needed to clarify the
[00:07:39.300]mechanism behind any change
[00:07:41.700]Beyond my own research.
[00:07:42.700]There's a massive MDH CURE's
[00:07:44.300]community that is set to dedicate
[00:07:45.500]years to the long journey
[00:07:47.600]to understand this life-sustaining
[00:07:49.700]enzyme.

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Exploration of Malate Dehydrogenase Structure and Functionality

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