Functional Analysis of Enzymes Involved in D-amino Acid Biosynthesis in Mycobacteria as Targets for Potential Therapeutics

Tim Kaftan, Olivia Taylor, and Alexander Belashchenko Author

08/04/2020 Added

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This is the video summarizing our summer research

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[00:00:03.479]Hello out research title is
[00:00:05.887]"Functional Analysis of
[00:00:07.377]Enzymes involved in D-amino Acid
[00:00:09.487]Biosynthesis in Mycobacteria as
[00:00:11.475]Targets for Potential Therapeutics"
[00:00:14.545]My name is Timothy Kaftan. I'm
[00:00:16.535]Olivia Taylor
[00:00:17.609]and our other research partner
[00:00:19.291]is Alexander Belashchenko
[00:00:21.646]Our research advisor is Dr. Raul Barletta
[00:00:27.818]Mycobacteria are fairly
[00:00:29.410]nonstandard because
[00:00:30.490]they can’t be stained with a standard
[00:00:32.364]Gram stain process but an acid fast stain
[00:00:34.424]procedure shows they are Gram-positive
[00:00:36.844]and this is particularly important
[00:00:38.179]for our research because it means they
[00:00:40.193]have a thick layer of peptidoglycan which
[00:00:42.543]is the component our lab is targeting.
[00:00:44.733]M. tuberculosis is the mycobacteria
[00:00:46.728]responsible for TB and thus our research
[00:00:48.727]would attempt to break down its
[00:00:51.057]peptidoglycan wall to counter it however
[00:00:54.139], because M. smegmatis mirrors many
[00:00:56.392]similar properties and is safer
[00:00:57.942]to work with in a lab because
[00:00:59.386]it isn’t pathogenic we actually
[00:01:01.186]use M. smegmatis strains
[00:01:02.477]in place of M. tuberculosis.
[00:01:04.132]Mycobacteria are aerobic and but they can
[00:01:06.714]actually survive and grow in low
[00:01:08.788]oxygen conditions and take on a rod or
[00:01:11.110]bacilli shape. The World Health
[00:01:13.867]Organization reported that in 2015
[00:01:16.039]there were 10.4 million people living
[00:01:18.051]with tuberculosis, 1.4 million deaths
[00:01:22.117]were attributed to tuberculosis,
[00:01:24.110]and 6.4 million new cases were diagnosed.
[00:01:27.794]Furthermore, this poses an even larger
[00:01:30.046]potential risk because currently
[00:01:31.922]a third of the world’s global population
[00:01:34.358]is infected with the bacteria that
[00:01:36.302]causes tuberculosis, Mycobacterium
[00:01:38.495]tuberculosis, which means that
[00:01:40.109]although these people may not currently
[00:01:42.521]have the condition, they may develop
[00:01:44.780]it in the future. Additionally, there has
[00:01:46.471]been a recent emergence of drug-resistant
[00:01:48.000]strain so it’s important to find new
[00:01:50.166]targets when designing drugs which is
[00:01:52.121]what our lab is currently doing.
[00:01:55.570]Previously published research from our
[00:01:57.764]lab titled "Isolation and
[00:01:59.383]Characterization of M. smegmatis
[00:02:01.773]murl Mutants Suggest the Existence of
[00:02:04.180]a New Pathway of D-glutamate
[00:02:06.087]Biosynthesis" by Govardhan Rathnalah
[00:02:09.061]and Fatimah Barnawl tells us that
[00:02:14.961]inhibtion of known enzymes does not
[00:02:18.864]prevent cell growth. This slide shows the
[00:02:22.496]biosynthesis of peptidoglycan
[00:02:24.662]as can be seen here, l-alanine
[00:02:26.830]is converting to d-alanine
[00:02:29.177]via alanine racemase
[00:02:30.988]what was shown in previous
[00:02:33.636]research was that when alanine racemase,
[00:02:36.104]or ALR, is inhibited cell growth
[00:02:38.045]can still continue
[00:02:41.435]This slide highlights the
[00:02:44.387]stereoisomer conversions between
[00:02:46.800]amino acids glutamine and
[00:02:48.050]alanine as well as the
[00:02:49.271]reversable reactions
[00:02:50.344]converting alanine to glutamate.
[00:02:51.993]This summer our lab focused on
[00:02:53.562]the conversion
[00:02:54.262]between d-alanine and d-glutamate
[00:02:55.773]via the enzyme d-alanine transaminase
[00:02:57.441]The following videos explain the
[00:03:02.412]protocol that was conducted
[00:03:07.984]Yesterday the cells were innoculated
[00:03:10.450]and allowed to grow for
[00:03:13.136]16 to 24 hours. Today we
[00:03:15.587]transferred these cells to a
[00:03:18.057]centrifuge tube and spun for
[00:03:20.774]ten minutes at 10,000 RPMs
[00:03:24.496]Now we're going to remove the
[00:03:26.078]supernatant and resuspend
[00:03:27.315]the cells in 100 milli-molar tris
[00:03:31.392]After we resuspend the cells we will
[00:03:33.687]transfer them to these fast prep
[00:03:36.190]tubes which contain very small minute
[00:03:39.158]beads and they will be mechanically lysed
[00:03:41.260]After we've ran the tubes through the fast prep
[00:03:48.631]machine the cells were then lysed we next
[00:03:52.751]put them back in the centrifuge so that we
[00:03:55.269]could remove the cell
[00:03:56.837]metabolites from the debris
[00:03:58.938]and now we're going to pipet
[00:04:01.207]them into dialysis bags to
[00:04:03.208]further seperate the large and
[00:04:05.276]small metabolites so we can get the
[00:04:07.444]metabolites that we want to look at
[00:04:09.562]for our assay.
[00:04:13.815]On day 3 of the protocol we
[00:04:15.350]prepare reactions by adding PLP,
[00:04:17.617]d-glutamate, d-alanine, and ketoacid
[00:04:20.463]A byproduct of the reactions is
[00:04:22.626]pyruvate which is the measured
[00:04:24.451]concentration in our results--see above
[00:04:27.378]High pyruvate content tells
[00:04:29.738]us that the reaction is in fact
[00:04:32.540]taking place and strongly supports the
[00:04:34.805]existence of a d-alanine transaminase
[00:04:36.268]enzyme. We plan to continue
[00:04:39.254]our research in the fall
[00:04:40.405]due to covid-19 we
[00:04:41.505]were unable to complete all of
[00:04:43.156]our planned experiments
[00:04:45.525]once said experiments are completed
[00:04:47.352]we plan to publish our results

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Functional Analysis of Enzymes Involved in D-amino Acid Biosynthesis in Mycobacteria as Targets for Potential Therapeutics

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