Characterization of Substrate Channeling in SmPutA

Tessa Hoffman Author

07/27/2021 Added

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This study examines substrate channeling within the context of the enzyme proline utilization A (PutA). A wild type and a channel blocked mutant are compared, and suggestions for further research are provided.

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[00:00:01.100]Hello. My name is Tessa Hoffman,
[00:00:02.820]and I'm a member of Nebraska's 2021 Redox Biology REU.
[00:00:06.880]This summer, I've been working in the Becker lab,
[00:00:09.030]studying the "Characterization of Substrate Channeling in SmPutA."
[00:00:14.030]The Becker lab broadly researches proline metabolism.
[00:00:16.910]My work focuses specifically on the proline catabolic pathway,
[00:00:20.217]in which proline is oxidized to glutamate.
[00:00:22.788]In the first step of this pathway,
[00:00:24.710]proline is converted to the intermediate P5C by proline dehydrogenase,
[00:00:28.990]or PRODH.
[00:00:30.440]This oxidation also involves the reduction of FAD to FADH2.
[00:00:35.420]P5C then undergoes a nonenzymatic hydrolysis
[00:00:38.420]to form glutamic semialdehyde,
[00:00:40.540]or GSA.
[00:00:42.054]Lastly, GSA is converted to the final product, glutamate,
[00:00:45.610]by P5C dehydrogenase,
[00:00:47.390]or P5CDH.
[00:00:49.010]A reduction of NAD+ to NADH also occurs in this final step.
[00:00:53.846]My research studied this pathway within the context of
[00:00:57.100]the enzyme proline utilization A, or PutA.
[00:01:00.879]PutAs are unique because they combine the activity of both PRODH and P5CDH
[00:01:05.694]into one enzyme with two active sites.
[00:01:08.714]In this representation of PutA,
[00:01:10.580]we can see the PRODH domain on the left,
[00:01:12.600]with the P5CDH domain on the right.
[00:01:15.530]The P5C or GSA intermediate travels from the PRODH to the
[00:01:20.180]P5CDH active site through a physical tunnel within the enzyme,
[00:01:24.430]shown here in gray.
[00:01:26.060]This passing of an intermediate from one active site directly to another
[00:01:29.671]is an ideal example of substrate channeling.
[00:01:33.680]Previous studies have identified various key benefits of substrate channeling.
[00:01:37.910]In some circumstances, channeling can result in an enhanced reaction rate,
[00:01:41.840]meaning that the final product is formed more quickly.
[00:01:44.840]Channeling can also offer a cell protection from the intermediate,
[00:01:47.870]which could be toxic if leaked out of the enzyme.
[00:01:50.660]On the other hand,
[00:01:51.740]protection is provided to the intermediate to keep it from degrading.
[00:01:55.860]Finally, substrate channeling can help minimize participation
[00:01:58.890]of the intermediate in other pathways.
[00:02:01.320]For example, my protein, PutA,
[00:02:03.350]takes the intermediate, P5C, and converts it to glutamate.
[00:02:07.410]However, if P5C were free in the cell,
[00:02:10.080]other enzymes might take P5C and convert it back to proline,
[00:02:13.860]or, it could go another direction into the ornithine pathway.
[00:02:17.980]Because PutA channels the intermediate from one site to another,
[00:02:21.060]it ensures that P5C gets made into glutamate.
[00:02:26.040]For the design of my project,
[00:02:27.540]I studied the PutA protein specifically from
[00:02:29.865]the bacteria Sinorhizobium meliloti,
[00:02:31.850]shortened as SmPutA.
[00:02:34.593]To examine channeling in this protein,
[00:02:36.591]I used a couple of SmPutA variants.
[00:02:39.240]The first of these is the wild type protein–
[00:02:41.298]meaning that it is completely natural with no alterations.
[00:02:44.670]The second is an asparagine to tyrosine mutation in the 999th position,
[00:02:49.590]AKA N999Y.
[00:02:52.410]This mutation replaces an asparagine in the protein's tunnel with a tyrosine.
[00:02:57.490]Because of the difference in size,
[00:02:59.170]the mutation blocks the tunnel within the enzyme,
[00:03:01.390]and prevents intermediates from
[00:03:02.860]channeling from the PRODH active site to the P5CDH active site.
[00:03:08.200]The first step in my methods involved transforming each SmPutA
[00:03:11.390]gene into a cell line; in my case, E. coli cells.
[00:03:14.980]This allowed the cells to produce a protein
[00:03:16.870]not naturally found in their genome.
[00:03:18.750]Next, the cells were induced to overexpress SmPutA
[00:03:22.200]so that there would be an excess
[00:03:23.620]of the protein in the cells to be used in studies.
[00:03:26.590]SmPutA was then isolated from the cells
[00:03:29.119]and purified with a nickel affinity column,
[00:03:31.763]and finally, the purified proteins were used in a variety
[00:03:34.647]of substrate channeling studies.
[00:03:37.672]Now, I will highlight the key findings of these studies.
[00:03:41.470]This first study shown is a channeling assay.
[00:03:43.880]Here, the protein is provided with proline and the production of the final product,
[00:03:47.849]glutamate, is determined;
[00:03:49.542]but because glutamate is difficult to track,
[00:03:52.120]we monitored the production of NADH instead,
[00:03:54.700]which is directly proportional to the amount of glutamate produced.
[00:03:58.151]The wild type SmPutA showed a curve like what would be expected:
[00:04:02.080]a buildup of glutamate over time,
[00:04:03.820]eventually leveling out as the system reaches equilibrium.
[00:04:06.815]However, the N999Y mutant showed no channeling activity,
[00:04:10.684]meaning that, provided with proline,
[00:04:12.504]the protein was not producing glutamate.
[00:04:15.190]The next studies examine this relationship,
[00:04:17.650]providing insight into the cause behind
[00:04:19.566]this lack of activity seen in the N999Y mutant.
[00:04:24.610]For this PRODH activity assay, the protein is again provided with proline.
[00:04:29.040]However, this time the production of P5C is monitored.
[00:04:32.870]This isolates the study to only the PRODH active site,
[00:04:36.020]removing the variable of the P5CDH active site.
[00:04:39.691]Again, because P5C is difficult to monitor directly,
[00:04:43.440]the reduction of FAD to FADH2 is tracked instead.
[00:04:47.550]Both the wild type and the N999Y mutant followed a typical Michaelis-Menten curve,
[00:04:52.230]meaning that the rate of reaction increased with increasing substrate levels
[00:04:56.460]until eventually leveling off as the protein became saturated with substrate.
[00:05:00.840]Because the N999Y mutant showed results very similar to those of the wild type in
[00:05:04.913]the PRODH activity assay,
[00:05:06.900]we can conclude that the PRODH active site of the mutant is not responsible
[00:05:10.770]for its lack of channeling activity.
[00:05:14.040]This next study is a P5CDH activity assay.
[00:05:17.401]In this case, the intermediate P5C is provided to the protein,
[00:05:21.420]then the amount of glutamate produced is monitored.
[00:05:24.120]This assay is complimentary to the previous study.
[00:05:26.652]This time, the activity of the P5CDH active site
[00:05:29.792]is isolated from activity of the PRODH active site.
[00:05:33.405]As before, NADH is directly tracked instead of glutamate,
[00:05:36.844]and, like the PRODH assay,
[00:05:38.970]the wild type protein showed the expected Michaelis-Menten kinetics.
[00:05:42.383]However, the N999Y mutant showed almost no activity,
[00:05:46.380]Although there was some slight variation in reaction rates,
[00:05:49.260]the activities are all extremely low when compared to those of the wild type.
[00:05:53.430]The inactivity in the P5CDH domain of the mutant is likely the cause of
[00:05:57.487]the inactivity seen earlier in the channeling activity assay.
[00:06:01.430]It is unknown exactly why this channel blocking mutation causes inactivity in
[00:06:05.513]the P5CDH active site,
[00:06:07.369]but we do have some hypotheses.
[00:06:09.397]First, it is possible that the N999Y mutation blocks the entrance
[00:06:13.175]to the P5CDH active site,
[00:06:15.420]which would stop the intermediate from being converted to glutamate.
[00:06:18.830]Another hypothesis is that the N999Y mutation somehow inactivated the
[00:06:22.880]P5CDH domain through damaging structural changes.
[00:06:27.548]To summarize, wild-type SmPutA was found to follow the expected
[00:06:31.202]Michaelis-Menten patterns in all studies.
[00:06:33.562]The channel blocked N999Y mutant
[00:06:35.760]also followed a Michaelis-Menten curve in the PRODH activity assay.
[00:06:39.651]However, little to no P5CDH activity was seen in this mutant.
[00:06:44.540]This was evident in the lack of channeling activity
[00:06:46.746]and also in the limited P5CDH function.
[00:06:50.260]Overall, these results allow us to conclude that the tunnel blockage
[00:06:53.219]is somehow leading to a lack of P5CDH function.
[00:06:56.970]In the future, these studies should be repeated to verify
[00:06:59.616]and strengthen these findings.
[00:07:01.428]Later, it may be helpful to do further structural analysis of SmPutA,
[00:07:05.523]specifically regarding the entrance location
[00:07:07.774]for the intermediate to the P5CDH active site,
[00:07:10.526]and also to verify whether or not the N999Y mutation
[00:07:14.191]has caused unexpected structural changes,
[00:07:16.730]which have inactivated the P5CDH domain.
[00:07:19.880]Eventually, this work could be replicated with PutAs from other bacteria to see
[00:07:23.720]if these findings hold true across other genomes.
[00:07:26.647]Finally, I would like to thank everyone
[00:07:28.513]who has been involved with my work throughout the summer.
[00:07:31.030]I'd like to extend a special thank you
[00:07:32.840]to my PI, Dr. Becker, for allowing me to learn and work in his lab;
[00:07:36.736]to my mentor, Yizi Mao, for teaching and providing guidance on my project;
[00:07:41.087]to all the other members in the Becker lab,
[00:07:43.007]for their support and encouragement throughout the summer;
[00:07:45.545]to the University of Nebraska-Lincoln,
[00:07:47.321]the Redox Biology Center,
[00:07:48.818]and the National Science Foundation,
[00:07:50.367]for providing funding and resources;
[00:07:52.269]and to the entire staff involved with the Summer Undergraduate Research Program,
[00:07:56.514]for helping make this summer possible.
[00:07:58.296]Thank you.

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Characterization of Substrate Channeling in SmPutA

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