Invited responses to my nylonase research and the question of “New Proteins Without God’s Help”

Susumo Ohno (who coined the term “junkDNA”) published a paper in 1984 through the National Academy of Sciences that was used by the NCSE, Ken Miller and Dennis Venema to claim “proteins can evolve without God’s help”. At the request of John Sanford, a courtesy associate research professor at Cornell, I was recruited to write a paper to refute Ohno’s evolutionary hypothesis on nylonases. I wrote it under John’s guidance based on his intuitions about genetics, his life-long specialty of 40 years and for which he became famous as attested by the fact he is one of the few geneticists who had their work featured in the Smithsonian National Museum of American History.

The actual paper is now in review, but it is not intended to be published in any journal, but will be released in a variety of channels shortly. It is hoped the material can be used by others to actually create papers that enter peer review. The motivation for releasing the paper in this way is to counter Venema’s book while it is still hot off the press.

The paper is being also published in this way so as to invite discussion since it isn’t intended to be considered a completely vetted product but one that welcomes improvement. That said, the sentiment among IDists and creationists who’ve seen the drafts is that paper has utterly discredited Ohno’s claims and thus the claims of the NCSE, Ken Miller and Dennis Venema connected to Ohno’s hypothesis of nylonase evolution.

Because the draft paper is a massive VJTorley-sized paper (15 pages in the main section and almost 80 pages of supplemental material) I’m establishing this thread at TSZ to invite review of the some of the themes from the paper that I’m releasing on the nylonase.XYZ website piece by piece in a format adapted for a website.

As I release each webpage, I’ll post in the comment section at TSZ a link to the newly constructed page so as to invite commentary on that page. Thank you in advance to all those willing to participate in this public review of my research on nylonase evolution.

NOTES:

1. “proteins can evolve without God’s help” is a paraphrase of the title of an NCSE article New Proteins Without God’s Help. Thwaites at the NCSE basically framed the debate over nylonase evolution in this way:

We’ve been trying to explain all this to the protein “experts” at ICR for the last seven years. We have told them that new proteins could indeed form from the random ordering of amino acids. We have warned them that their calculations were based on faulty assumptions and soon someone would document the natural formation of a new protein from the random association of amino acids.

Now it has happened! Not one, but two, new proteins have been discovered. In all probability new proteins are forming by this process all the time, but this seems to be the first documentation of this phenomenon. The newly discovered proteins are enzymes that break down some of the byproducts produced during nylon manufacture. Since nylon first came into commercial production in 1940, we know that the new enzymes have formed since that time.

2. The nylonase.XYZ website is under construction, so don’t click around the website too much yet. In the comment section at TSZ I will link to individual pages of the website that can be reviewed individually.

3. The first comments by me at TSZ will be more technical, not for the beginners regarding Ohno’s work. The beginner and introductory stuff will be added later to the website.

174 thoughts on “Invited responses to my nylonase research and the question of “New Proteins Without God’s Help”

  1. I don’t know what to make of this except that Banks seem to be predatory lenders toward African Americans especially. Now, bankers have notoriously been left wingers in contrast to small business owners who are generally right wingers. The so called subprime mortgages were an instrument supposedly to help poor black families. It turns out they’ve been a mechanism to pillage and enslave and plunder black families. Left-wing “charity” and “affirmative action” and “racial equality” shows it’s true colors again:

    http://prospect.org/article/staggering-loss-black-wealth-due-subprime-scandal-continues-unabated

    As the wealthiest black-majority county in the United States, Prince George’s has long represented the pinnacle of black success. The county’s median household income is 73,568—a full20,000 more than the median household income of the United States as a whole. Only 7.1 percent of U.S. firms are black-owned, but in Prince George’s that number stands at a whopping 54.5 percent.

    A full 29.5 percent of people over the age of 25 hold bachelor’s degrees—slightly higher than the 28.5 percent rate for all persons in the United States. Known colloquially as just P.G., the county is filled with lawyers, entrepreneurs, teachers, and federal employees. In popular lore, Prince George’s was proof that, while blacks still lagged behind in education, wealth and employment, the black community was finally catching up.

    But in 2007, the bursting of the housing bubble triggered an economic recession that rippled throughout the global economy. For years, the housing market had been booming; in 2007 the U.S. median price for a house hit a record high of 247,900. By 2009, though, that number had fallen to216,700. For Prince George’s County, however, the decline was much more stark. In 2009 the median price for a house dropped by nearly 100,000, from343,000 to just $245,000.

    Although the foreclosure crisis left no part of the country untouched, in the Washington, D.C., area—which, overall, weathered the crisis well—Prince George’s County bore the brunt. The reason? Subprime lending.

    Specifically targeted for subprime loans among the minority demographic were black women. Women of color are the most likely to receive subprime loans while white men are the least likely; the disparity grows with income levels. Compared to white men earning the same level of income, black women earning less than the area median income are two and a half times more likely to receive subprime. Upper-income black women were nearly five times more likely to receive subprime purchase mortgages than upper-income white men.

    From 2005 to 2009, the net worth of black households declined by 53 percent while the net worth of white households declined by 16 percent, according to Social & Demographic Trends researchers at the Pew Research Center. At the peak of the housing boom, 49 percent of blacks owned homes while the same measure for whites hovered around 75 percent.

    Historically, the wealth gap between whites and blacks can be traced back to the ability to own land; for a number of years blacks were prohibited from owning land, and once homeownership became the primary way to own property black people were often barred from that, too.

    Now who is the Piece of Doodoo who advocated pushing subprime mortgages on Black Americans. Well, look here:

    With landmark lawsuit, Barack Obama pushed banks to give subprime loans to Chicago’s African-Americans

    President Barack Obama was a pioneering contributor to the national subprime real estate bubble, and roughly half of the 186 African-American clients in his landmark 1995 mortgage discrimination lawsuit against Citibank have since gone bankrupt or received foreclosure notices.

    Well done, champion of Black America, Barrack Obama.

  2. There is BLAST and PSI-BLAST. PSI-BLAST uses the hidden markov model and will reasonably detect structural similarities. I just ran the part of Ohno’s sequence that he claims is due to the frame shift. That means, I had to take out the part that would not be affected by the frameshift namely the methionine start and several amino acids thereafter. The snipped part is:

    MGYIDLSAPVAMIVSGGLYYLFTRRGYTFGDT

    this leaves the part Ohno said must have existed. It is very telling there is no similarity hit even with the more relaxed PSI-BLAST hidden markov models:

    RERTFHRPAPRQVSRSRGRGADTRQLAG

    GPAQPLGLRPPGRAAAHGGGLPARPGDARGARRAARRARDAAPRSRAAARGDLHRRIPRA

    ARLRGPRRVLPGGFRTRRPSPADERLEVAVRHGRRRADRRGAHRSRAARHRVCTRARGLR

    LRRALRAAGARHADLDRLQRGLRRSGLGGADPRSLRRLAHAARRGPRRHLRVPHHPPRRR

    RHRRVPVLLGEHRRARLDRRAGHRSALRRSALHVPVGEARRRSGCDHHGRPDRLRLRERG

    RLLHRAGSRTRGPHDARRRRRSRRTGRIAGLGGKRAGRRLPRSHDRRGFHLRIPRGQLHA

    PVVVHGQRARQRERHRHPRPEPLARSAHRLGDRQALVVARSRHPALARAAERDPARRQPC

    PRRGVGG

    After all said and done, not even PSI BLAST could pick anything up.

    In contrast, when I ran PSI-BLAST on the KI72 NYLB form, I got 10,000 hits at least!!!!

    Use the accession number for the KI72 NYLB enzyme: P07061.1

    Then use the PSI-BLAST Option, expand the algorithm parameters section and allow up to 10,000 “max target sequences”.

    I should note, the WORST hit listed had an e-value of e^-9, and had the following entry:

    6-aminohexanoate hydrolase [Maledivibacter halophilus]
    69.7 69.7 81% 2e-09 20% WP_079490286.1

    This classification is of “6-aminohexanoate hydrolase” is a nylonase.

    There is an interesting issue with NylB-PRIMEs. They seem to have a fold all their own. What I mean is, you can blastp plain vanilla NylB for KI72 but will miss hits on NylB-primes that are listed in UniProt.

    For example, blast will not find sequence homology of KI72’s NylB’ with this NylB’:

    https://www.ncbi.nlm.nih.gov/protein/950320365

  3. stcordova,

    The paper you referenced is from the National Institute of Health? A government agency? Did you list that as you being a benefactors of where your money is supposed to go in your amazingly stupid tax plan?

  4. I created test phylogeny of a sampling of the NylB’s found in 193 organisms using :

    http://phylogeny.lirmm.fr/phylo_cgi/index.cgi

    The data I put through


    >NylB KI72
    mnarstgqhp arypgaaage ptldswqeap hnrwafarlg ellptaavsr rdpatpaepv
    vrldalatrl pdleqrleet ctdaflvlrg sevlaeyyra gfapddrhll msvskslcgt
    vvgalidegr idpaqpvtey vpelagsvyd gpsvlqvldm qisidynedy vdpasevqth
    drsagwrtrr dgdpadtyef lttlrgdggt gefqycsant dvlawiverv tglryveals
    tylwakldad rdatitvdqt gfgfanggvs ctardlarvg rmmldggvap ggrvvsqgwv
    esvlaggsre amtdegftsa fpegsytrqw wctgnergnv sgigihgqnl wldprtdsvi
    vklsswpdpd trhwhglqsg illdvsrald av

    >NylB Cupriavidus necator (strain ATCC 43291 / DSM 13513 / N-1) (Ralstonia eutropha)
    MQAQTVTRRT GFGRSRAWLA VALLVAPMSV ALAQGTGQPL VVAEDEVMQG
    FPPPPDKQVS RGSGLRPPYM RWAFRHAREM SPTAGIRHAS QPLALAGQPG
    TELDGTTFAV AGKTARLADY LRDTHTDGFI VLHQGKVVYE RYLAGFGPYQ
    PHIWASMTKS VTGLLAAMLV EEGKLDPQAR LAQYVPELAG NPFGEATVQQ
    NLDMEVPVGY PEGLPPDLGL FGAVGIVPRK ADAPDTIYDF LKVVHATGGR
    DEVGVWYYQN GSPEAVAWAL RRITGKSWAQ LVTERLWSRF ADDDAYTQVD
    RQGTEMASGG MNSTLRDAAR FAEAVRRAAA GDASGGISPA AVRIALQPAG
    NQARFARGNT MPGRDGYGYR NYWFQRNDGD GSIEASGRFG QKIYINPARG
    LTVVKFSANP DGAARATSAA GVRKRDDPGR ALESAEAMVA AALAIHRAVS

    >NylB_1 Bacillus cereus
    MKLKKSPLLL LIITFIFVIT GLGFTYLKHN KTTPSKNNVT KKNWLDDPYL
    RWSYTHMKEF TLINDVKNNP DQIARFPSAL QNLDDFAVQR RFGSTTPLKE
    LLDDNKTDAF VVVHNGQLVY ERYFNGYNES EPHGMASLAK VFTGAIIQSL
    AEENRIDLEK TADTYIKELK NTPFGKATLQ QLMDMQVSVE YPTHGYEHPA
    LENQDAQLYL ASNILPRGKN YDGPMKIYDM LQEAKETAPP GSVFSYNNGS
    TETLAWIIRT ITGKSLAENV SERIWSQIGM EENAYYVTDE TKIEQASAGL
    NATARDMARF GQLLLNNGEY NGKQILPSSI TEDIKNVQQG ELAIGPGASI
    SYHNQWWIPH NEQGAFEVLG SYGQTLYIDP KANMVIVHFS SNATPSNEIH
    SVYSNMYIDI AHHLEKLPQ

    >NylB_2 Bacillus cereus
    MDFKQLENKF EKKKVNTFLV YQKGELTTEY YKTPECANNL YKINSITKSI
    VSILIGIAIG KGYINDIHTP ITKWILNVPK EKSELTIYHL LTMTTGEDWK
    EFGNGVVFPN DFVESENWVQ YILAKPIIEE PATKMNYNSG SSHLLSYIIQ
    KATGMTTEQF AKRYLFDPLE ITEYEWQQDP QGIHVGGFGM KMKSKDLLKL
    GILCLQNGYW QGNEIVSSKW LGESSTALFE TYEHVGAYGY HWWVLNNERF
    HIPYCMYFAM GYGGQYIVII PQLEVVAVIS SHMPKRGLVP LKLFIEHVQE
    NYKFG

  5. I crossposted this somewhere in Common Design vs. Common Descent thread.
    http://theskepticalzone.com/wp/common-design-vs-common-descent/comment-page-55/#comment-197151

    At the bottom is an example of an alignment that supposedly is comparable to the hidden Markov Models, it is MUSCLE. I used it to align 4 nylonase genes, 3 of which have evidence of actual nylonase enzymatic activity, not to mention, the genes are have the same name, namely, NylB (for nylonase B).

    The funny capitalization is the result of me pulling the data from UNIRPOT vs. GenBank.

    I used the MUSCLE alignment on the Clustal Server:
    https://www.ebi.ac.uk/Tools/msa/muscle/

    >NylB KI72
    mnarstgqhp arypgaaage ptldswqeap hnrwafarlg ellptaavsr rdpatpaepv
    vrldalatrl pdleqrleet ctdaflvlrg sevlaeyyra gfapddrhll msvskslcgt
    vvgalidegr idpaqpvtey vpelagsvyd gpsvlqvldm qisidynedy vdpasevqth
    drsagwrtrr dgdpadtyef lttlrgdggt gefqycsant dvlawiverv tglryveals
    tylwakldad rdatitvdqt gfgfanggvs ctardlarvg rmmldggvap ggrvvsqgwv
    esvlaggsre amtdegftsa fpegsytrqw wctgnergnv sgigihgqnl wldprtdsvi
    vklsswpdpd trhwhglqsg illdvsrald av

    >NylB Cupriavidus necator (strain ATCC 43291 / DSM 13513 / N-1) (Ralstonia eutropha)
    MQAQTVTRRT GFGRSRAWLA VALLVAPMSV ALAQGTGQPL VVAEDEVMQG
    FPPPPDKQVS RGSGLRPPYM RWAFRHAREM SPTAGIRHAS QPLALAGQPG
    TELDGTTFAV AGKTARLADY LRDTHTDGFI VLHQGKVVYE RYLAGFGPYQ
    PHIWASMTKS VTGLLAAMLV EEGKLDPQAR LAQYVPELAG NPFGEATVQQ
    NLDMEVPVGY PEGLPPDLGL FGAVGIVPRK ADAPDTIYDF LKVVHATGGR
    DEVGVWYYQN GSPEAVAWAL RRITGKSWAQ LVTERLWSRF ADDDAYTQVD
    RQGTEMASGG MNSTLRDAAR FAEAVRRAAA GDASGGISPA AVRIALQPAG
    NQARFARGNT MPGRDGYGYR NYWFQRNDGD GSIEASGRFG QKIYINPARG
    LTVVKFSANP DGAARATSAA GVRKRDDPGR ALESAEAMVA AALAIHRAVS

    >NylB_1 Bacillus cereus
    MKLKKSPLLL LIITFIFVIT GLGFTYLKHN KTTPSKNNVT KKNWLDDPYL
    RWSYTHMKEF TLINDVKNNP DQIARFPSAL QNLDDFAVQR RFGSTTPLKE
    LLDDNKTDAF VVVHNGQLVY ERYFNGYNES EPHGMASLAK VFTGAIIQSL
    AEENRIDLEK TADTYIKELK NTPFGKATLQ QLMDMQVSVE YPTHGYEHPA
    LENQDAQLYL ASNILPRGKN YDGPMKIYDM LQEAKETAPP GSVFSYNNGS
    TETLAWIIRT ITGKSLAENV SERIWSQIGM EENAYYVTDE TKIEQASAGL
    NATARDMARF GQLLLNNGEY NGKQILPSSI TEDIKNVQQG ELAIGPGASI
    SYHNQWWIPH NEQGAFEVLG SYGQTLYIDP KANMVIVHFS SNATPSNEIH
    SVYSNMYIDI AHHLEKLPQ

    >NylB_2 Bacillus cereus
    MDFKQLENKF EKKKVNTFLV YQKGELTTEY YKTPECANNL YKINSITKSI
    VSILIGIAIG KGYINDIHTP ITKWILNVPK EKSELTIYHL LTMTTGEDWK
    EFGNGVVFPN DFVESENWVQ YILAKPIIEE PATKMNYNSG SSHLLSYIIQ
    KATGMTTEQF AKRYLFDPLE ITEYEWQQDP QGIHVGGFGM KMKSKDLLKL
    GILCLQNGYW QGNEIVSSKW LGESSTALFE TYEHVGAYGY HWWVLNNERF
    HIPYCMYFAM GYGGQYIVII PQLEVVAVIS SHMPKRGLVP LKLFIEHVQE
    NYKFG

    The resulting aligment is depicted below which can be reproduced easily from the above data. It echoes the point that about 12% of the sequences actually align for the same protein.

  6. Here is a computationally generated “phylogeny” of 48 nylB proteins. This only 1/4 of the organisms I found with NylB.

    Ken Miller should be embarrassed for promoting Ohno’s frame shift hypothesis in light of the act Ohno’s hypothesis can’t account for all the varieties of NylB in this diagram alone. The most parsimonious explanation is that NylB pre-existed 1935 and Ohno’s frame shift didn’t create the NylB form.

    [NOTE: one can zoom their browser to see finer details]

  7. Given the sheer number of different NylB enzymes, combined with their apparent huge sequence divergence, there must be an incredible number of possible linear oligomer hydrolases possible. Even a superficial glance at your alignment shows only a rough conservation of a chemical motif.

    Probably, almost all of those NylB enzymes are actually not really adapted to work on the nylon waste oligomer, but are divergent enzymes from a superfamily of enzymes that degrade a whole host of antibiotics.

  8. stcordova: There is an interesting issue with NylB-PRIMEs. They seem to have a fold all their own. What I mean is, you can blastp plain vanilla NylB for KI72 but will miss hits on NylB-primes that are listed in UniProt.

    That’s sounds very strange to me. Isn’t the KI172 NylB and NylB-prime only different from each other by like 12 amino acids?

    And how do you distinguish a NylB from a NylB-prime anyway? Aren’t they all just NylB versions? The original nomenclature was suggested only because, at the time, NylB and NylB-prime was known from the pOAD2 plasmid harbored in Flavobacterium Sp. KI172. And either of them is a duplicate of the other, very similar to each other (differing only by 12 out of 392 amino acids).

    I would find it quite fantastic that an enzyme that exists in so many different versions (12% sequence similarity or maybe even less), can fold into a different structure entirely with a mere 12 amino acid substitutions yet continue to catalyze the same reactions at a relatively tiny loss in rate of substrate turnover.

    Please clarify what you’re doing on Uniprot. Are you saying that if you blast the Flavobacterium Sp. KI172 NylB enzyme amino acid sequence, that you do NOT get any hits for the Flavobacterium Sp. KI172 NylB-prime sequence?

  9. Rumraket,

    Thank you for your response. A couple things, as you know, I have been about the only creationist who said the improbability of a protein fold for some function may not be as remote as some creationists claim. The data I provided confirm this rather pointedly, and hence my caution was warranted.

    Given the sheer number of different NylB enzymes, combined with their apparent huge sequence divergence, there must be an incredible number of possible linear oligomer hydrolases possible. Even a superficial glance at your alignment shows only a rough conservation of a chemical motif.

    No kidding! I mean, if trypsin can be a nylonase, it seems relatively easy to me to make a nylonse. That was the conclusion Dr. Sanford and I made….

    I suppose we could re-check Doug Axe’s work using the same approach with Beta Lactamases. 🙂

    It’s worth pointing out NylB is annotated as in the Beta Lactamase family. What do I think about protein improbability? For enzymatic activity, it depends on the reaction being catalyzed, some reactions are easier than others, and it’s usually not on or off, but how fast. NylB-prime will catalyze a some nylonase reactions that NylB can, but 1000 times slower. Same with NylA and NylC (NylB is linear oligomers, NylA is Cyclic Dimers, NylC is Cyclic Oligomers, NylB-Prime is uknown what it does).

    Given this data, I may strongly re-consider whether we cite Doug Axe’s work at all in our re-write of the nylonase paper.

    The reason? From a previous comment by me:

    should note, the WORST hit listed had an e-value of e^-9, and had the following entry:

    6-aminohexanoate hydrolase [Maledivibacter halophilus]
    69.7 69.7 81% 2e-09 20% WP_079490286.1

    An e-value of e^-09 isn’t exactly astronomically remote!!!! And given the BLAST e-values are database dependent, it might even be a bigger e-value, like (guestimate) e^-05. Heck, who knows…..

  10. That’s sounds very strange to me. Isn’t the KI172 NylB and NylB-prime only different from each other by like 12 amino acids?

    Actually 46 amino acids in KI72, but that is about 11-12% of the 392 residues. The number is different in other creatures.

    I independently confirmed the UNIPROT numbers on a limited sample size by cross checking GenBank.

    But first, to see what I’m seeing, it is rather easy. Go to UNIPROT and type “nylB” in the search box. You’ll get a flood of hits on NylB and NylB’. So the gene prediction software that was used to annotate genes in GenBank seems to somehow know the difference between NylB and NylB’.

    I’m going to load some the cleaned up data points on the Nylonase website after I get the OK from Dr. Sanford. In the mean time I’ll provide some it piecemeal here.

    Ok for starters here are apparently the critical residues. Nylonases appear to be Serine Hydrolases.

    https://en.wikipedia.org/wiki/Serine_hydrolase

    Superfamilies of serine hydrolases includes:
    Serine proteases, including trypsin, chymotrypsin, and subtilisin
    Extracellular lipases, including pancreatic lipase, hepatic lipase, gastric lipase, endothelial lipase, and lipoprotein lipase

    My paper with Dr. Sanford listed examples of Proteases and Lipases that can act as nylonases!

    I uploaded an image file which you can hopefully click on and examine below. You should be able to see the column where the Serine position is conserved (letter “S”) the lysine position (letter “K”) the Isoleucine/Lysine postion (letter “I” and “L”).

    I think the NylB-PRIME has a different positioning of these critical residues. I guess I could look into it. 🙂

    http://theskepticalzone.com/wp/wp-content/uploads/2017/10/serine_position.png

  11. stcordova: I have been about the only creationist who said the improbability of a protein fold for some function may not be as remote as some creationists claim.

    I hope no one missed this.

  12. If one does a Clustal MUSCLE aligment with the following sequences, one will see that 7 positions are 100% conserved. ROUGHLY speaking, for a NylB for to be hit by random chance is 1 out of 20^7 or 1.3 billion. I doubt Ohno’s frame shift could hit this. If we include the partially conserved motifs, the probability is even more remote. Granted these aren’t Doug Axe numbers, but they aren’t highly probable numbers either.

    Here are the sequences which interested readers can cut and paste. When I get the green light, I’ll put stuff like this up on the nylonase website.

    In the meantime you can cut and paste the sequence below into the Clustal MUSCLE website here:
    https://www.ebi.ac.uk/Tools/msa/muscle/

    >Flavobacterium_K172
    MNARSTGQHP ARYPGAAAGE PTLDSWQEAP HNRWAFARLG ELLPTAAVSR
    RDPATPAEPV VRLDALATRL PDLEQRLEET CTDAFLVLRG SEVLAEYYRA
    GFAPDDRHLL MSVSKSLCGT VVGALIDEGR IDPAQPVTEY VPELAGSVYD
    GPSVLQVLDM QISIDYNEDY VDPASEVQTH DRSAGWRTRR DGDPADTYEF
    LTTLRGDGGT GEFQYCSANT DVLAWIVERV TGLRYVEALS TYLWAKLDAD
    RDATITVDQT GFGFANGGVS CTARDLARVG RMMLDGGVAP GGRVVSQGWV
    ESVLAGGSRE AMTDEGFTSA FPEGSYTRQW WCTGNERGNV SGIGIHGQNL
    WLDPRTDSVI VKLSSWPDPD TRHWHGLQSG ILLDVSRALD AV

    >Bacillus_thuringiensis
    MKLKKSPLLL LILTFIFVIT GLGFTYFKQN KTTPSKHNVT KENWLNDPYL
    RWSYTHMKEF TLVNNVKNNP DQIAHFPSAL QNLDDFAVER RFGNTTPLKK
    LLDDNKTDAF VVVHNGQLVY ERYFNGYKQN EPHGMASLAK VFTGAIIQSL
    AEEKRIDLEK TADTYIKELK NTPFGNATLQ QLMDMQVSAE YPTHGYEQPG
    LENQDAQLYL ASNILPRGKN YDGPMKIYDM LREAEETAPP GSSFSYNNGS
    TETLAWIIKT ITGRSLAENV SERIWSQIGM EENAYYVTDE TKVEQASAGL
    NATARDMARF GQLLLNNGEY NGKQILPSSI TEDIKNVQEG ELAIGPGASI
    SYHNQWWIPH NEQGAFEVLG SYGQTLYIDP KANMVIAHFS SNATPSNEIH
    SVYSNMYVDI AHYLEKLPQ

    >Cupriavidus_necator
    MQAQTVTRRT GFGRSRAWLA VALLVAPMSV ALAQGTGQPL VVAEDEVMQG
    FPPPPDKQVS RGSGLRPPYM RWAFRHAREM SPTAGIRHAS QPLALAGQPG
    TELDGTTFAV AGKTARLADY LRDTHTDGFI VLHQGKVVYE RYLAGFGPYQ
    PHIWASMTKS VTGLLAAMLV EEGKLDPQAR LAQYVPELAG NPFGEATVQQ
    NLDMEVPVGY PEGLPPDLGL FGAVGIVPRK ADAPDTIYDF LKVVHATGGR
    DEVGVWYYQN GSPEAVAWAL RRITGKSWAQ LVTERLWSRF ADDDAYTQVD
    RQGTEMASGG MNSTLRDAAR FAEAVRRAAA GDASGGISPA AVRIALQPAG
    NQARFARGNT MPGRDGYGYR NYWFQRNDGD GSIEASGRFG QKIYINPARG
    LTVVKFSANP DGAARATSAA GVRKRDDPGR ALESAEAMVA AALAIHRAVS

    >Bacillus_cereus_NylB_1
    MKLKKSPLLL LIITFIFVIT GLGFTYLKHN KTTPSKNNVT KKNWLDDPYL
    RWSYTHMKEF TLINDVKNNP DQIARFPSAL QNLDDFAVQR RFGSTTPLKE
    LLDDNKTDAF VVVHNGQLVY ERYFNGYNES EPHGMASLAK VFTGAIIQSL
    AEENRIDLEK TADTYIKELK NTPFGKATLQ QLMDMQVSVE YPTHGYEHPA
    LENQDAQLYL ASNILPRGKN YDGPMKIYDM LQEAKETAPP GSVFSYNNGS
    TETLAWIIRT ITGKSLAENV SERIWSQIGM EENAYYVTDE TKIEQASAGL
    NATARDMARF GQLLLNNGEY NGKQILPSSI TEDIKNVQQG ELAIGPGASI
    SYHNQWWIPH NEQGAFEVLG SYGQTLYIDP KANMVIVHFS SNATPSNEIH
    SVYSNMYIDI AHHLEKLPQ

    >Bacilus_cereus_NylB_2
    MDFKQLENKF EKKKVNTFLV YQKGELTTEY YKTPECANNL YKINSITKSI
    VSILIGIAIG KGYINDIHTP ITKWILNVPK EKSELTIYHL LTMTTGEDWK
    EFGNGVVFPN DFVESENWVQ YILAKPIIEE PATKMNYNSG SSHLLSYIIQ
    KATGMTTEQF AKRYLFDPLE ITEYEWQQDP QGIHVGGFGM KMKSKDLLKL
    GILCLQNGYW QGNEIVSSKW LGESSTALFE TYEHVGAYGY HWWVLNNERF
    HIPYCMYFAM GYGGQYIVII PQLEVVAVIS SHMPKRGLVP LKLFIEHVQE
    NYKFG

    >Arthrobacter_Rue61a
    MTTTGAHPPR YVGAAGQPTV DSWQEGPHNR WTFAHLGEML PTASVTRHFP
    AAPAAATERL DALAVPGLSQ RLEESYTDAF LVLHRNNVIA EYYRPGFAPD
    DLHLVMSISK SMCGLVVGAL VDSGDIVTSD RVVHYVPELA GSAYDGPTIQ
    HVLDMAIHLN YSEDYMDPAS EVQTHDRSSG WRTRRDGDPQ DTYEFLTTLT
    GNGTVGRFQY CSANTDVLAW IIERVTGLRY SEALSKYLWS KLDADRDATI
    TVDSSGFGFA NGGVSCTARD LARVGRLMLD GGVGPAGRVV SEGWVRSILA
    GGDREAMADA SFTGIHPQGS YTRQWWCTGN ERGNVTGIGI YGQYLWIDPA
    TDTVVVQLST WPEPDSGHLH ELQNQLLLDV SRSLDQPLAS QEIPTEETAQ
    ESKETAA

    >Streptococcus_pneumoniae_nylB_2
    MKLKKSPLLL LILTFIFVIT GLGFTYFKQN KTTPSKHNVT KENWLNDPYL
    RWSYTHMKEF TLVNNVKNNP DQIAHFPSAL QNLDDFAVER RFGNTTPLKK
    LLDDNKTDAF VVVHNGQLVY ERYFNGYKQN EPHGMASLAK VFTGAIIQSL
    AEENRIDLEK TADTYIKELK NTPFGNATLQ QLMDMQVSAE YPTHGYEQPG
    LENQDAQLYL ASNILPRGKN YDGPMKIYDM LREAEETAPP GSAFSYNNGS
    TETLAWIIRT ITGKSLAENV SERIWSQIGM EENAYYVTDE TKIEQASAGL
    NATARDMAKF GQLLLNNGEY NGKQILPSSI TEDIKNVQEG ELAISPGASI
    SYHNQWWIPH NEQGAFEVLG SYGQTLYIDP KANMVIVHFS SNAMPSNEIH
    STYSNMYIDI AHHLEKLPQ

    >Shinella_DD12
    MMKTFKDEHG FTRADVTLAN WRTAPYSRWT FQNVRDLVPT AVIAAEVPAA
    EAPLPSDAFL DAAMETGLAG AASARAFLDF AHTDAFVMMR RGEIVAEYYA
    PHANPDAPHL VFSISKSLTA VVSGILEADG LIDPDRPVAD YLPEAKGSVY
    GDCTYRDVLD MRVSLDFEEA YLDPHGAFAR YRRAMLWNPP EPNIAPETLA
    SFLVTLEKAE RPHGGPFYYA SPNADLLGVV IERVTGARFA DLTSDLLWKP
    MGAKGNANIT VDAIGTPRTA GGVSMTARDL ARLGELLRNH GVRNGRQIVP
    EGWIRDMQEN GDREAWQQGR NVDLPNGRYR SQWYQSGEAD GAFCAIGIHG
    QWLYVDPSTE TVIVKLSSQP EPLGDEENQD NFTFFRALCR RAI

    >Streptococcus_pneumoniae_nylB
    MKLKKSPLLL LILTFIFVIT GLGFTYFKQN KTTPSKHNVT KENWLNDPYL
    RWSYTHMKEF TLVNNVKNNP DQIAHFPSAL QNLDDFAVER RFGNTTPLKK
    LLDDNKTDAF VVVHNGQLVY ERYFNGYKQN EPHGMASLAK VFTGAIIQSL
    AEEKRIDLEK TADTYIKELK NTPFGNATLQ QLMDMQVSAE YPTHGYEQPG
    LENQDAQLYL ASNILPRGKN YDGPMKIYDM LREAEETAPP GSSFSYNNGS
    TETLAWIIRT ITGKSLAENV SERIWSQIGM EENAYYVTDE TKVEQASAGL
    NATARDMARF GQLLLNNGEY NGKQILPSSI TEDIKNVQEG ELAIGPGASI
    SYHNQWWIPH NEQGACEVLG SYGQTLYIDP KANMVIAHFS SNATPSNEIH
    SVYSNMYVAI AHYLEKLPQ

    >E_coli
    MIRKPLALAL ILAALPAAAM AQHCGSLTLD VCPTPYDQTL PAAKDMLSWD
    QTSRVIGFRN DYRNYAGDVF RHGASTPLER AEKQLTDARY TLNGHTWNLQ
    DYLKRENVSG MLVLKDGKVA WKYLAEGNTD TTLWTSRSVG KSVVSTLVGI
    AIQQGKIHSL DDLITVYEPE LKGTAWDGVT LKQLIQHTSG VEWNEDYTDP
    QSHFARLTQC EAHPGVYACV RKIVTGLARQ HPAGEQWSYS SGGAWLLGDI
    LERATGMSLA AWLEQALWQP AGMAHDGVWH AYQQGKHDVG AHGFNATLED
    WGRFGEFVAR DGRLSNGKQL VPAGWFDQAA SWTKALNSVS AAHPEGIYGY
    QWWNNAIPAN AQNVQPTPQE GLKGSLWALG IYGQVIMVNR AEHLVIVQWS
    TWPQAEPSFN AQPLEAALMY SAIARELR

    >Chryseobacterium_MOF25P
    MIVIIYLMAF LAAVAVLFYL FGYTYIFNGI SKTYLRGKTS ANIDDGKLFT
    SNIIHTTKPV LWDEHSDYNK KDLPKIIVDD LTHSNTASFL VIKDGKLLHE
    QYWNGYNELS KTNSFSMAKA VTVMLFGKAL EEGKIKNIDA SFSEFYDEFK
    NKPLGKEVSL KHLAQMESGL NWDENYKNPF LPNARAYYGT SLMKATFSRT
    FKEKPGERFE YQSGSTQLLG FAVKKAVNQS LSSYLSEKFW IPLGMEQNAT
    WSVDESGMEK TYCCIHSNSR DFAKLGQLFL DDGKVGDQQI LNLDFIEQMR
    TPTKKSDEIY GMGLWINNDN PIKHYYFLGL QGQYIIIIPE YKMVIVRTGS
    YNNLPKTDRG RPDQVKFLVN ETVKLFA

    >Ruegeria_atlantica_nylB_4
    MRTLMKWVLR IVLMLVLAAV VVGVWKREEI QRLMAVNSLF SEEKIIHNFS
    HMDDAFLTVD LPRGEGATFE LPYGPGFELP EGTDKWVEDR AVTSLLVMQD
    GQIRFEEYYL GTTPEDRRIS WSVAKSYLSA LFGILMAEGA IDSLDDPVVK
    YAPKLQGTAY DGATIRNVLN MASGVTFDED YLDYNSDINR MGRVVALGGE
    LDDFAASLNE TFVEPGETWQ YVSIDTHVIG MVVRGATDRS VADLLTEKII
    EPLGLERDGY YVTDGAGVAF VLGGLNFTTR DFARFGQMIL QNGEHNGQQI
    VPAEWIAESV FPSAPTEAGE IGYGYQWWIP VGAHEGEFLA RGVYGQYIYF
    DQPRGVVIVS TGADRSFRDD GVNETNIEMF RTIAQSL

    >Ruegeria_atlantica_nylB_5
    MRTLMKWVLR IVLMLVLAAV VVGVWKREEI QRLMAVNSLF SEEKIIHNFS
    HMDDAFLTVD LPRGEGATFE LPYGPGFDLP EGTDKWVEDR AVTSLLVMQD
    GQIRFEEYYL GTAPEDRRIS WSVAKSYLSA LFGILMAEGA IDSLDDPVVK
    YAPKLQGTAY DGATIRNVLN MASGVTFDED YLDYSSDINR MGRVVALGGE
    LDDFTASLNE TFVEPGETWQ YVSIDTHVIG MIVRGATDRS VADLLTEKII
    EPLGLERDGY YVTDGAGVAF VLGGLNFTTR DFARFGQMIL QNGEHNGQQI
    VPAEWIAESV FPSAPTEAGE IGYGYQWWIP VGAHEGEFLA RGVYGQYIYF
    DQPRGVVIVS TGADRSFRDD GINETNIEMF RTIAQSL

    >Parabacteroides_distasonis
    MKNDSNNRVP ANKHYLSRRV GWIVFIFMLG IGGYLALPSN YYLRRALIHL
    LPKIDQYPIF ENRVVKAGSP RPWELSEAYN TKSIPERYLP RFEELGTVAY
    VIIQDNRLLF EQYWEDYSPE SHSNSFSMAK SIVSLAIGCA IDDGFIRDVD
    QPVSDFFPEF KGYDGKALTL RHLLTMSAGV DFDEAYSSPF SPTTQLYYGD
    DLQEIAFGMK EIDEPGVNFI YQSGVTQLLG FIVEKATGEK LSDYVSRKLW
    TPMHAEESAL WSLDRKDGME KAYCCFNSNA RDFARFGQLL LNNGQWDDRQ
    LISPAYLAEA TSPDTRLVYK DLGKPNHCYG FQYWILDYKG MKIPYMRGIL
    GQYVFTLPEK NAVIVRLGHK RSDTYTADQH YPDDINTWLD AAMDLLQ

    >Cupriavidus_taiwanensis_DSM_17343_nylB2
    MQAQAKSRGK GLRRSRGWRT VVPLVALMVA PAGVALAQGA GQPAAVAESE
    VMQGFPPPPD KQVGRGTGLR PPYMRWAFRH AREMSPTVGI RHASLPLALP
    GQSAPELDAV GFTVAGQTVR VADYLRDTHT DGFIVLHQGR IVYERYLAGF
    DPHQPHIWAS MTKSVTGLLA AMLVEEGRLD PQARLAQYVP ELAGNPFGEA
    TVQQNLDMEV PVGYPQALPP DLGLFGAVGI VPRKADAPDT IYDFLKVVHA
    TGSAGEGPDG GVWYYQNGSP EAVAWALRRV TGKRWADLVT ERLWSRFADD
    DAYTQVDRQG TEMASGGMNS TLRDAARFAE TVRRAAAGDA TVGISPKAVR
    IALQPASNQA RFARGNTTAG RDGYGYRNYW FQRNDGDGSI EASGRFGQKI
    YINPARELTV VKFSASPDGA ARATSAAAVR KRDDPARALE SSEAMVAAAH
    ALLRAASR

    >Klebsiella_pneumoniae
    MIRKPLALAL ILAALPAAAM AQHCGSLTLD VCPTPYDQTL PAAKDMLSWD
    QTSRVIGFRN DYRNYIGDVF RHGASTPLER AEKQLTDARY TLNGHTWNLQ
    DYLKRENVSG MLVLKDGKVA WKYLAEGNTD TTLWTSRSVG KSVVSTLVGI
    AIQQGKIHSL DDLITVYEPE LKGTAWDGVT LKQLIQHTSG VEWNEDYTDP
    QSHFARLTQC EAHPGAYACV RKIVTGLARQ HPAGEQWSYS SGGAWLLGDI
    LERSTGMSLA AWLEQALWQP AGMAHDGVWH AYQQGKHDVG AHGFNATLED
    WGRFGEFVAR DGRLSNGKQL VPAGWFDQAA SWTKALNSVS AAHPEGIYGY
    QWWNNAIPAN AQNVQPTPQE GLKGSLWALG IYGQVIMVNR AEHLVIVQWS
    TWPQAEPSFN AQPLEAALMY SAIARELR

    >Marinobacter_hydrocarbonoclasticus_ATCC_49840
    MAALNKEKTM NSAVILGFAL SLALGGTAVA QEATQLSAPD STPDALGLMQ
    GFPPAPDKTI RFTDPDYFAF PKLRWTVCHF RELMPTTVVR NGSEGVSELP
    VDMDAGIEGV EFLPLGGKAS MTWRDAFDAN YTDGILVLHQ GRVVYERYDG
    CLDEHTLHGA MSVTKSLTGL LGEVLVAEGK LDETALVGDI IPELGRSAFG
    DATVRQVLDM TTALDYSEDY SDPDAEVWTY ARAGSPYLVS EQREGPRSYF
    DYLKTVRKDG EHGEAFGYKT INSDVVGWLI ARTTGVSVAD YFSERIWSKI
    GAEREAFYTV DSIGTPFAGG GFNATLRDMA RIGLLVLNEG KWAGEQIIPA
    QAIASIRNGG DRAVFAKAGY ELLDGWSYRG MWWVSHDDHG AFAARGVHGQ
    TIWIDPAADM VIVRFASNPV AGNAANDPTS LPAYRAVADY LKSQERGAGP
    AGGP

    >Altererythrobacter_namhicola_nylB_2
    MSRRSRSPNP LARLLPALLA AAVLAGCGSD GPPPEPPLPE SALEAVVESP
    GVEREKLARK IDALFTAEGI GETRAVIVMH RGEVVAERYA EGFGPGTRFI
    GWSLSKTVTG LMIGALVAEG QLALDESPPI PRWQRSGDPR GEITIRQLLQ
    MRSGLRHAEA SDPVYESPEV RMMFLDGRDD MAAWAQAQPL EHEPGAEFDY
    STNTSVILAD VAARVLAPGA GPDARREAVA GFLESRLATP MGAPSFTGEF
    DAQGTMLAGS NIWATARDWA KLGELLRNGG SKGGVQLVPR SWVDFMRRPS
    PRASDYGAHL WLNRASGNDR TVLWPAQGPD TAFAAVGHLG QYVVVSPEQG
    LTVVRLGKST NEERDLMVPL LAELFALYPL T

    >Altererythrobacter_namhicola_nylB_3
    MKVAIKNMLL AAGGAMALTA CSAQTIDTTP NQPDVMASMP GGDVAGLQTA
    ATQVLFWDDA TRADRFRRME DFFPGLVVAP SPDPRDLTFG EPMPAAAIEA
    LDSYIAKGDT AGILVLQGGQ VRYENYGLGM GPQDRWTSFS MAKSVTSTLA
    GAALKDGFIT SLQDPVSQYI PGLRGSAYDD VTIEQLLTMT SGVAWNEDYT
    DPASDVARMF AVEPVAGEAQ VVTYMKTLPR EAPAGEKFVY KTGETNLIGV
    LVEKATGQSL AAYAKSKIVD PAGFQKAMFW QTDLTGGNVG GCCLALTLQD
    YGRLGQWTLE GAQGTVPEGW LAAAGSAQTD FGNGFGYGYQ WWTYPQGSYG
    AVGIFGQTIT LLPEQDAVIV VLGNWPRATG GDLNQGRLQL VNTVTLAMGR
    E

    >Sphingobium_EP60837
    MKLERKRVKQ IGAAAAILSL MGAGALAVRA ADPEPRYHVA ADAGVNEEDL
    RAAIDPLFDG DEDVGETRAL VVMHRGEIVA ERYAPGFGPE TKLLGWSMGK
    SVTAVLVGLM VADGRLALDS PVPVAAWSQQ GDPRGRITLR QLLAMTSGLD
    HVEDEEPLAQ ADTVRMLFTD GAQDMSAFAE AKPLAHAPGS HFSYSTGSTL
    ILTDLMARML TNNADPDARR QAMQTFIDGR LKIPAGLKSL TAEYDAAGTM
    IGGSFLHMTA RDYAQFGELL RNHGRGPNGH QIVPEKWVDF MRTPSRRNPA
    YGAHLWLNRE SEESVLMPGD APQSLFGCAG HGGQYILISP GQALTVVRIG
    MSPDKEQRAA LKRRLAKLMR LFAA

    >Vibrio_scophthalmi
    MKLKLLTVLA SISTVSLLAS ANANTMYSVP ADAKNQVTLS NWTLPEHNHW
    SFQNASIHPN VMVPRDGNVS VLPEKLDPTI SELKFEYSGT TYTVHEAMTN
    DRTDGYVVVK DGQIVHEEYF GTFNAKSQHM WASSTKSMIG QAVGLLVEQG
    KIDPNKTVET YIDELKDSHL GKQTVRTILN MTSALDYSED YANITPGTVH
    FEYFRRLGFI PAYDLMALDP TKDDTPRGLL EFASYIDQNP ELKPNEIFEY
    HSPNVDIAGW LVARVSGQPL QDFIAQNIWY KLGVEHDALF MTDMTYTPVA
    TGGFATTLRD FARVGIAVAN NGAYNNQQVF SEAWIKDTFN LTDDEKQHVN
    RSVYKDKGSS GYDEWLEGYK NYLWVHDSEK GIATFRGVFG QNLYINQEKN
    LVIATFSSAN SASNVARVTN LPRMAAFEAI ANTY

    >Altererythrobacter_atlanticus_nylB_2
    MTRPFKINRF PSRAAALLPA LLPLLLVSAC KDEGPPAPPP VPPEVLATVA
    EEPGTDREDL ARAVDALFTR DDIGETRALI VMHAGEVAAE RYGGEYGPET
    KFLGWSMSKT VTGVLMGMMV ADGRLRLDDS PPIPSWQRAG DPRGEITLRQ
    LLQMRSGLRH QEKAEPVYTS DEVRMMFLDG RDDMAAWAEA QPLEHEPGRE
    FDYSTASATI LSDIGARVLA PEGSADKRQA AMDDFLHARL GVPLGMKSLT
    AEYDRAGTMV GGSMIWATAP DWARFGEFLR HGGSVKGAQI VPRGWIDFMK
    SESPRAPDYG ATLWLNRDSG TDREMLFPES GPESLFAAIG HLGQYVLVSP
    SQKLTVVRLG KTDEEDRAAL VDALAEIVAL YP

    >Altererythrobacter_dongtanensis
    MRHPASLIAA LAALPAPLLL LAACSSQPPA PDPLTKEALA AVRADPGAPT
    EQLAREIDDL FANEGLGETR AVVVMHAGEL AAERYAAGYG PETRFVSWSM
    AKTVTATMIG LLVADGRLTL DETPPIPRWR RAGDPRGEIT LRQLLQMRSG
    LRHTESGDPP YESSEVRMLF LDGRDDMATF AESQPLEAEP GREFEYSSNT
    TVILADIAAR VLTRSTDPDV RRKAVADYLQ TRLFGPLGMT SMVPEFDRAG
    TLVGGSLMHG TARDWARFGE FLRNKGSYRG TQVVPRRWVE FMTTPSPRSG
    HYGAQTWLNR DPPEGNDPLF ADRGPKSLFA MIGHMGQYVL VAPDRKLTVV
    RLGHSDREER PPMLQELADV VALYPGT

  13. A fragment showing the 2 of the 7 conserved positions (highlighted by asterisks at the bottom of the diagram), Serine and Lysine (letter “K”), is shown. It think it almost astronomically improbably Ohno’s random frame shift would land on the NylB architecture. This proves Ohno, Ken Miller and Dennis Venema wrong. Miller used Ohno’s claims to argue against Stephen Meyer. Miller loses this round.

    You can zoom in here, but shown below as well.
    http://theskepticalzone.com/wp/wp-content/uploads/2017/10/serine_position_v3.png

  14. stcordova: A fragment showing the 2 of the 7 conserved positions (highlighted by asterisks at the bottom of the diagram), Serine and Lysine (letter “K”), is shown. It think it almost astronomically improbably Ohno’s random frame shift would land on the NylB architecture. This proves Ohno, Ken Miller and Dennis Venema wrong. Miller used Ohno’s claims to argue against Stephen Meyer. Miller loses this round.

    You should completely drop this conservation argument as a reason why the Ohno frameshift didn’t happen as it’s outright fallacious reasoning. The fact that some amino acid is highly conserved isn’t what makes Ohno’s frameshift hypothesis unlikely. In light of data Ohno didn’t have, we now know this enzyme exists in many different species and is extremely divergent. That means the alternative reading frame Ohno gleaned from the pOAD2 plasmid is pretty much just a statistical artifact of the particular NylB enzyme it happens to encode.

    Besides, that Lysine isn’t completely conserved. In a picture you showed earlier, Microbacterium has an R (Arginine) in that same position.

  15. stcordova: If one does a Clustal MUSCLE aligment with the following sequences, one will see that 7 positions are 100% conserved. ROUGHLY speaking, for a NylB for to be hit by random chance is 1 out of 20^7 or 1.3 billion. I doubt Ohno’s frame shift could hit this. If we include the partially conserved motifs, the probability is even more remote.

    Your reasoning here is nonsensical. Whether any positions are conserved or not, or how many of them there are, is completely besides the point wrt whether we should infer a recent frameshift mutation created NylB and NylB-prime.

    What argues against it is the fact that linear nylon waste oligomer hydrolysis-capable enzymes seem to be rather ubiquitous and highly divergent. Which means however that whole family of enzymes originated, it must have happened many hundreds of millions of years ago. And with that level of sequence divergence between all these different enzymes, it would simply not be possible to infer an ancestral, shifted reading frame from so long ago. It would have been erased long ago, leaving no evidence to support the inference that such a frameshift mutation ever took place.

    So the correct argument against Ohno is: There is no evidence the whole of the NylB family was created by a frameshift mutation. It’s not a probability argument that has anything to do with the number of conserved-or-not residues, or motifs, in a comparison between different NylB enzymes.

  16. Rumraket: Besides, that Lysine isn’t completely conserved. In a picture you showed earlier, Microbacterium has an R (Arginine) in that same position.

    .. and a G (Glycine) in place of the S (Serine).

    Notice something peculiar about those two substitutions? They are chemically quite alike. It is plausible that Serine could be replaced by Glycine, while Lysine could be replaced with Arginine. They are close in size and polarity (G – S, K – R).

  17. Rumraket,

    I thought the microbacterium might have wrong gene prediction, it missed a few other spots, so I removed it. What do you think? Should I keep it. As far as I know it, like the most, are unreviewed non-experimentally tested gene predictions. So I pulled it. Was that wrong?

    Thanks in advance.

  18. Well I wonder how many of these predicted NylBs actually are NylBs. In place of actually testing these enzymes it seems that what you get are just proteins calculated to be likely to adopt a similar fold.

    If that is so I see no reason to exclude it, after all for all we know that enzyme could be capable of linear oligomer hydrolysis catalysis too. And it still seems to roughly correspond to the same overall motif.

  19. Rumraket:

    Your reasoning here is nonsensical.

    Sorry for the late reply, and I especially feel bad since this was one of the high quality criticisms, so much so I withdraw what I said. I won’t post it in that form on the website and most certainly not in the re-write of my paper.

    Thank you for you comment and criticism, that was a good one.

  20. Rumraket:

    Well I wonder how many of these predicted NylBs actually are NylBs. In place of actually testing these enzymes it seems that what you get are just proteins calculated to be likely to adopt a similar fold.

    If that is so I see no reason to exclude it, after all for all we know that enzyme could be capable of linear oligomer hydrolysis catalysis too. And it still seems to roughly correspond to the same overall motif.

    Thank you again for you comment.

    Because this was an on-going concern throughout development of the paper, I finally consulted someone at the NIH Monday evening over the topic. He worked with Masotoshi Nei and has published with Eugene Koonin, and has published phylogenies, so he’s as good as anyone…

    I told him about my concern about mis-labeling in the process of gene annotation, and he said, “absolutely you should be skeptical….the quality of the naming is only as good as the bio-informatics person in the lab.” And that’s freaking scary since I saw a researcher release a genome in gen bank and he even had less clue than me about gene annotation! He got some guy to use some software to do the annotation. I was brand new at the time to this whole world and I was appalled to find people release annotations all the time just based on the genome, not on mRNA transcripts, much much less on actually mass spectrometry or what ever assays on the actual proteins!

    I remember the whole class laughing when I asked, “can we take a cell and sequence all the proteins.” My dumb question of the day. They then explained the expense of using Mass-Spec to do it exhaustively….

    So…..

    I learned one other important thing at the NIH. The PSSM matrix.

    https://en.wikipedia.org/wiki/Position_weight_matrix

    There are PSSMs that are supposedly qualified via X-Ray crystallography. I suppose they form the matrix of proteins sequences that have the same fold or domain as identified by X-Ray crystallography.

    As far as NylB, one can go to GenBank and enter this accession number for KI72’s NylB: P07061.1

    then click on “idenfity conserved domains”. It’s that easy. 🙂

    And voila, you get a description of the PSSM profile NylB is homologous to:
    “CubicO group peptidase, beta-lactamase class C family [Defense mechanisms]; ”

    Thus, even though the MUSCLE alignment looks kind of poor, we can get a scoring for its similarity to a known domain. KI72 score 131 bits, about 10^-39 improbability of a match by chance to this particular Beta-Lactamase fold. Who knows, maybe NylB’s have their own canonical fold. I don’t know.

    For the reader’s benefit, I provided a link that you can zoom in on the PSSM matrix comparison for NyLB.

    http://theskepticalzone.com/wp/wp-content/uploads/2017/10/beta_lactamase_pssm.png

    I must admit, I first had no idea why I kept seeing the word “AmpC” in the databases for NylB. Why do they call the regions “AmpC” and not “NylB” in many gen bank entries. Now I know. “AmpC” is the name of the PSSM of the conserved fold that NylB has the strongest match for in the PSSM library.

    All this to say, I have to look up some of the NylB’s and see how well they line up to the AmpC PSSM.

    This also lends credence to a point you made here:
    http://theskepticalzone.com/wp/axe-enw-and-protein-sequence-space-again-again-again/

    A random peptide may not line up to a given fold (as evidenced by aligning to a PSSM), but it doesn’t mean it can align to another fold. The improbability calculations have to take this into account.

    Also, I don’t know how closely something must align to give it the AmpC fold. I guess that’s the question Doug Axe was trying to explore. I have no strong opinion of his numbers either way.

  21. FWIW,

    Doug Axe gave a figure of 10^-77 improbability of the beta lactamase fold.

    If this particular NylB has a beta-lactamase fold, and Negoro et all have X-Ray crystallography stuff on it, then maybe the data I shown has bearing on Doug’s numbers.

    The PSSM matrix score of KI72 NylB was 130.5 bits, or 1.9 x 10^-39. Does that mean a “random” peptide was shown to have a Beta Lactamase fold with only 1.9 x 10^-39 degree of specificity?

    An open question. But for the sake of completeness I mention it.

  22. What did my MUCLE diagram show is a conserved motif? Hmm…

    It looked something like S-X-X-K which means Serine-Something-Something-Lysine or equivalently Ser-X-X-Lys.

    from Negoro’s X-ray crystallography paper on nylonases:

    http://www.jbc.org/content/280/47/39644.full.pdf

    the “Ser-X-X-Lys motif”
    ….
    The
    present studies suggest a strategy to create new enzymes active toward
    various amide and ester compounds from an enzyme having “Ser-X-XLys”
    as a common active center.

    SHAZAM baby! I freaking nailed it. Woohoo!

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