The Structure and Iron-binding of a Divergent Isocyanide Hydratase in Bacillus

Nathlita Karnley Author

07/31/2021 Added

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Bacillus Isocyanide Hydratase which is an homolog from the enzyme called Isocyanide Hydratase was worked on. Two mutants of that homolog (BaICH) was worked on, to solve the protein structure because of the metal-binding located within the protein, and the lack of typical ICH activity.

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[00:00:00.870]Hello everyone. My name is Nathlita Karnley and
[00:00:03.630]I'm a senior at Fayetteville state university, majoring in biology.
[00:00:07.530]Today I would like to present on the structure and Iron-binding of a divergent
[00:00:10.740]Isocyanide Hydratase in bacillus.
[00:00:13.200]This research was proposed.
[00:00:14.190]By Dr. Wilson and Dr. Lin
[00:00:15.270]at the university of Nebraska from the department of biochemistry and the redox Biology center.
[00:00:19.080]Isocyanides are a
[00:00:22.970]Class of organic compounds that contain a triply bonded a carbon nitrogen
[00:00:26.330]functional group,
[00:00:27.380]which are small groups of atoms that exhibit a characteristic reactivity and often
[00:00:31.160]possess anti-microbial or transition metal bonding activities.
[00:00:35.980]Isocyanides can be described by two resonance structures. One,
[00:00:38.740]with a triple bond between the carbon-nitrogen
[00:00:42.110]double bond, which you can see below.
[00:00:45.380]In one resonance structure, an isocyanide has a formal positive charge
[00:00:48.410]on the nitrogen.
[00:00:49.490]And a formal negative charge on the carbon atom. In another resonance form, the
[00:00:54.050]carbon atom has a carbenoid character, and can act as an electrophile
[00:00:57.830]And it's single state.
[00:01:00.500]Isocyanide hydratase is an DJ-1 superfamily enzyme that hydrates isocyanides
[00:01:04.690]to yield the corresponding N-formamide.
[00:01:08.270]The DJ-1 super family is a structural and diverse group of proteins that are
[00:01:11.930]present in most organisms.
[00:01:14.840]ICH is commonly present in the genetic material of pseudomonad bacteria.
[00:01:19.070]That are likely to encounter isocyanides, proposing that the enzyme was needed to
[00:01:22.790]deactivate the toxic isocyanide natural products.
[00:01:27.440]Bacillus anthrax, is a rod-shaped, gram-positive bacterium, in which a divergent
[00:01:31.130]ICH
[00:01:31.450]homolog Ba-ICCH was reported from, this present study was undertaken to
[00:01:36.260]determine the structural binding-iron in Bacillus Isocyanide hydratase, and to
[00:01:40.280]determine the high resolution crystal structure in BaICH.
[00:01:44.990]I have been working with Dr. Lin and Dr. Wilson in the Wilson lab,
[00:01:48.110]and we have developed a design of data to understand about the functional
[00:01:51.560]diversity of ICH's, and structural diversity.
[00:01:54.380]of ICH's as a part of the the DJ-1 super family.
[00:01:58.910]in the ribbon diagram, of pseudomonas ICH,
[00:02:01.580]we see a dimeric enzyme, with each monomer colored differently.
[00:02:05.990]It has shown a location of a catalytic essential cysteine.
[00:02:08.450]Nucleophile labeled C 1 0 1 in pseudomonas.
[00:02:11.360]ICH and labeled C 1 0 3 and Ba-ICH
[00:02:17.390]in the figure below the ICH mechanism is depicted.
[00:02:20.450]And it shows that we have an active site cysteine nucleophile that attacks the
[00:02:23.900]carbenic atom of the Isocyanide that results in the intermediate.
[00:02:27.950]And then the intermediate is hydrolyzed by water to give the N-formamide,
[00:02:31.850]which is the product of the reaction. As we move down,
[00:02:36.200]we found that there is no activity out for BaICH and in the graph.
[00:02:39.890]It shows that BaICH, like the negative control Protein has no measurable activity,
[00:02:44.750]which was measured using para nitrophenyl isocyanide as a substrate,
[00:02:50.480]as we moved right up above, we depicted that protein purification,
[00:02:53.810]purifies the wild type protein.
[00:02:55.640]With a deep red color due to iron in the solution. While see C1 0 3 S and C 1 54S
[00:03:00.530]showed no color based on the ICP-MS. Result, known for inductively,
[00:03:05.260]coupled plasma, mass spectrometry,
[00:03:07.240]which measures elements at trace levels in biological fluids,
[00:03:10.690]we can measure and determine the content of metal in a sample,
[00:03:14.050]And in this sample,
[00:03:15.820]we can see that the color of the mutants are missing. Due to no iron binding
[00:03:21.130]Moving forward.
[00:03:21.790]We successfully crystallized all four cysteine mutants which crystallized
[00:03:26.410]nicely, and are all diffraction quality crystals, all four crystals are colorful.
[00:03:31.330]but the wild-type crystal is actually pink.
[00:03:33.820]While the rest of the crystals can be viewed as having unusual colors due to the
[00:03:37.660]view under polarized light. This isn't their true colorization.
[00:03:40.990]And with the absence of polarized light,
[00:03:42.910]the wild-type crystal is light pink and the rest of the crystals are actually clear.
[00:03:48.460]As we move right up above, the x-ray diffraction data
[00:03:52.300]collection at the Stanford synchrotron radiation lightsource,
[00:03:55.330]is shown and a cryoprotected crystal of C103S BaICH is displayed,
[00:03:59.890]displayed
[00:04:00.310]mounted in a loop and maintained at a hundred Kelvin before data
[00:04:04.420]collection,
[00:04:05.860]the white box called inset is showing and indicating where the x-ray beam light will hit
[00:04:09.970]the crystal. Down below we are shown the overall diffraction
[00:04:14.530]image, spotted and high resolution.
[00:04:16.870]And the top image is showing a single frame.
[00:04:18.870]of X-ray diffraction from C 1 0 3 S B a I C H.
[00:04:23.410]And the bottom image is an inset showing a large B axis of the
[00:04:27.760]crystal resulting in very close spaced spots. From this.
[00:04:32.290]We can conclude that Ba-IC H crystals have a large unit cell axis.
[00:04:37.930]And the overall conclusion of our studies is that Ba-ICH is a divergent member of
[00:04:42.520]the ICH family that does not have ICH activity in standard essays.
[00:04:47.410]The enzyme also purifies with deep red color due to iron- binding as determined using
[00:04:52.060]ICP-MS. Structural and biophysical studies
[00:04:55.840]are planned to determine the basis of iron binding by BaIC H.
[00:04:59.830]And the reason that it is inactive.
[00:05:03.790]I would like to thank Dr. Lin and
[00:05:05.230]Dr. Wilson for all their guidance and support during this research experience.
[00:05:08.830]And I would like to thank professor Berkowitz and Dr. Tiwari for the synthesis of the
[00:05:12.970]the P -N P I C substrate. I would like to thank Dr. Seravalli for the
[00:05:17.160]the ICP-MS analysis.
[00:05:19.030]And I like to thank the staff at beamline at SSRL for their assistance with
[00:05:23.170]X-ray data collection. Lastly,
[00:05:25.690]this work was supportive in part by the national science foundation and the
[00:05:29.050]national Institutes of Health.
[00:05:31.230]Thank you for listening. Thank you.

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The Structure and Iron-binding of a Divergent Isocyanide Hydratase in Bacillus

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