Intragenomic Biocatalyst Diversity
The family of Baeyer-Villiger Monooxygenases from Rhodococcus
jostii RHA1
Gideon Grogan, York Structural Biology Laboratory
BIOCATALYSIS
ENZYMES
cytP450Flavin-dependent
oxidoreductases
C-C bond lyases organosilanes
cytP450Flavin-dependent
oxidoreductases
C-C bond lyases organosilanes
ENZYMES
• Structure/mechanism – informed mutation
• Directed evolution - gene shuffling
- random point mutation and combinations thereof (CASTing)
• Metagenomics – amplify genes direct from environmental samples
• Genomics (1200 bacterial genomes)
- Amplify representative genes from different organisms
- Intragenomic complements
If an organism has accumulated n genes for an enzymatic reaction
is it reasonable to suppose that this small pool will provide a range of catalytic
diversity (i.e. organism has been equipped to do a number of jobs)
Generating and Exploiting Biocatalyst Diversity
Intragenomic complements
Intragenomic complements
Angew. Chem. Int. Ed. (2006) 45, 3534 - 3538
Baeyer-Villiger Monooxygenases
• Catalyse oxygen insertion adjacent to carbonyl groups
Malito et al. (2004) Proc. Natl Acad. Sci. USA, 101, 13157-13162
Baeyer-Villiger Monooxygenases – lots of different enzymes catalyse BV reaction
Rohr et al. (2009) Biochemistry, in press
Isupov and Lebedev (2008) Acta Cryst. (2008). D64, 90-98
Kim et al. (2005) Plant Cell, 17, 2397-2412
• 60 kDa ish enzymes that catalyse oxygen insertion (although may have
N-terminal extension)
• Are dependent on oxygen, FAD and NADPH
• Have two Rossman motifs (GXGXXG) for recognition of NADP and FAD
• Have a ‘Baeyer-Villiger monooxygenase’ motif FXGXXXHXXXWP/D
• Are hence broad sequence and structural homologs of CHMO and PAMO (structure)
Baeyer-Villiger Monooxygenases
----------------MMP--DYHALIVGAGFSGIGAAIKLD-RAGFSDYLVVEAGDGVG 41
VSIADTAAKPSTPSPANQPPVRTRAVIIGTGFSGLGMAIALQ-KQG-VDFVILEKADDVG 58
-----------------MTGRCPTVAVVGAGMSGMCVAITLL-SAGITDVCIYEKADDVG 42
------------------MTEHLDVVIVGAGISGVSAAWHLQDRCPTKSYAILEKRESMG 42
------------------MNQHFDVLIIGAGLSGIGTACHVTAEFPDKTIALLERRERLG 42
-------------MSVTPNAGCVDVVIVGAGISGLGAAYRIIERNPQLTYTILERRARIG 47
GTWHWNTYPGIAVDIPSFSYQFSFEQS----RHWSRTYAPGHELKAYAEHCVDKYGIRSR 97
GTWRDNTYPGCACDIPSHLYSFSFEPK----ADWKHLFSYWDEILGYLKGVTDKYGLRRY 114
GTWRDNTYPGLTCDVPSRLYQYSFAKN----PNWTQMFSRGGEIQDYLRGIAERYGLRHR 98
GTWDLFRYPGIRSDSDMYTLGFRFRP-----WTGRQAIADGKPILEYVKSTAAMYGIDRH 97
GTWDLFRYPGVRSDSDMFTFGYKFRP-----WRDVKVLADGASIRQYIADTATEFGVDEK 97
GTWDLFRYPGVRSDSSIFTLSFPYEP-----WTREEGIADGAHIREYLTDMAHKYGIDRH 102
IRLNTKVLAAEFDDEHSLWRVQ-----TDPGGEITARFLISACGILTVPK--LPDIDGVD 150
IEFNSLVDRGYWDDDECRWHVF-----TADGREYVAQFLISGAGALHIPS--FPEIAGRD 167
IRFGATVVSARFDDG--RWVLR-----TDSGTESTVDFLISATGVLHHPR--IPPIAGLD 149
IRFHHKVISADWSTAENRWTVH--IQSHGTLSALTCEFLFLCSGYYNYDEGYSPRFAGSE 155
IHYGLKVNTAEWSSRQCRWTVAGVHEATGETRTYTCDYLISCTGYYNYDAGYLPDFPGVH 157
IEFNSYVRAADWDSSTDTWTVT--FEQNGVHKHYRSRFVFFGSGYYNYDEGYTPDFGGIE 160
SFEGVTMHTARWDHTQDLTGKRVGIIGTGASAVQVIPEMAPIVS----HLTVFQRTPIWC 206
EFAGPAFHSAQWDHSIDLTGKRVAIVGTGASAIQIVPEIVGQVA----ELQLYQRTPPWV 223
DFRGTVFHSARWDHTVPLLGRRIAVIGTGSTGVQLVCGLAGVAG----KVTMFQRTAQWV 205
DFVGPIIHPQHWPEDLDYDAKNIVVIGSGATAVTLVPALADSG---AKHVTMLQRSPTYI 212
RFGGRCVHPQHWPEDLDYSGKKVVVIGSGATAVTLVPAMAGSNPGSAAHVTMLQRSPSYI 217
KFGGAVVHPQHWPEDLDYTGKKIVVIGSGATAVTLIPSLTDRA----EKVTMLQRSPTYL 216
FPKFDV-----PLPTAVRWAMRIPGGKAVHRLLSQAFVEATFPIAAHYF----------- 250
VPRTNE-----ELPVSLRRALRTVPG--LRALLRLGIYWAQEALAYGMT----------- 265
LPWPNP-----RYSKLARVFHRAFPC--LGSLAYKAYSLSFETFAVALS----------- 247
VSQPD-------RDGIAEKLNRWLPETMAYTAVRWKNVLRQAAVYSACQ----------- 254
FSLPA-------VDKISEVLGRFLPDRWVYEFGRRRNIAIQRKLYQACR----------- 259
ISASK-------YSTFAAVVRKALPPKTSHLIVRMYNALLEAVFWFLSR----------- 258
----------------AVFPLAKHMESAGRRYLRQQVHDPVVREQLTPRYAVGCKRPGFH 294
----------------KRPNTLKIIEAYAKYNIRRSVKDRELRRKLTPRYRIGCKRILNS 309
----------------NPGLHRKLVGAVCRASLRR-VRDPRLRRALTPDYEPMCKRLVMS 290
----------------KWP--RRMRKMFLSLIQRQLPEGYDVRKHFGPHYNPWDQRLCLV 296
----------------RWP--KLMRRLLLWEVRRRLGRSVDMSN-FTPNYLPWDERLCAV 300
----------------KTP--VFVKWLLRRTAIKNLPEGYDIETHFTPRYNPWDQRLCLI 300
N--TYLSTFNRDNVRLVTEPIDKITPTAVATTDGAS----HEIDVLVLATGFKVLDTDSI 348
S--TYYPAVADPKTELITDRIDRITHDGIVTADGTGREVFREADVIVYATGFHV--TDSY 365
G--GFYRAIQRDDVELVTAGIDHVEHRGIVTDDGVL----HEVDVIVLATGFDS--HAFF 342
PNGDLFRAIRHGKVEVVTDTIERFTATGIRLNSGRE----LPADIIITATGLNL------ 346
PNGDLFKTLASGAASVVTDQIETFTEKGILCKSGRE----IEADIIVTATGLNI------ 350
PDADLYNAITSGRAEVVTDHIDHFDATGIALKSGGH----LDADIIVTATGLQL------ 350
PTYAVTGTGGASLSRFWDEHRLQAYEGVSVPGYPNFFTVFGPYGYVGS-SYFALIETQAH 407
TYVQIKGRHGEDLVDRWNREGIGAHRGITVANMPNLFFLLGPNTGLGHNSVVFMIESQIH 425
RPMQLTGRDGIRIDDVWQ-DGPHAHQTVAIPGFPNFFMMLGPHSPVGNFPLTAVAESQAE 401
--QLFGGATATIDGQQVDITTTMAYKGMMLSGIPNMAYTVG----YTNASWTLKADLVSE 400
--QMLGGMRLIVDGAEYQLPEKMTYKGVLLENAPNLAWIIG----YTNASWTLKSDIAGA 404
--QALGGAAISLDGVEIDPRDRFVYKAHMLEDVPNLFWCVG----YTNASWTLRADMTAR 404
HIIRCLKRARRTGATRIEVTEEANARYFAEVMRRRHRQVFWQDSCRLANSYYFDKNGDVP 467
YVADAIAKCDRMGVQALAPTREAQDRFNQELQRRLAGSVWNSGGCR---SWYLDEHGKNT 482
HIVQWIKRWRHGEFDTMEPKSAATEAYNTVLRAAMPNTVWTTGCDS----WYLNKDGIPE 457
FVCRLLNYMDDNGFDTVVVERPGSDVEERP--FMEFTPGYVLRSLD-----ELPKQGSRT 453
YLCRLLRHMADNGY-TVATPRDAQDCALDVGMFDQLNSGYVKRGQD-----IMPRQGSKH 458
ATAKLLAHMAAHGHTRAAPHLGDEPMDEKP--SWDIQAGYVKRAPY-----ALPKSGTKR 457
SFEGVTMHTARWD
EFAGPAFHSAQWD
DFRGTVFHSARWD
DFVGPIIHPQHWP
RFGGRCVHPQHWP
KFGGAVVHPQHWP
* * *. :*
FXGXXXHXXWD/P
Baeyer-Villigerase motif
Baeyer-Villiger Monooxygenases from M. tuberculosis H37Rv
1 2 3 4 5 6 7
kDa
14
20
30
43
66
94
1. Markers 2. Total cell extract from E.coli B834 transformed with pDB1 (Rv0892),
3. pDB2 (Rv0565c), 4. pDB3 (Rv3854c), 5. pDB4 (Rv1393c), 6. pDB5 (Rv3049c),
7. pDB6 (Rv3083c)
Rv3049c
Baeyer-Villiger Monooxygenases from M. tuberculosis H37Rv
0
20
40
60
80
100
0 1 2 3 4
Time (h)
Co
nv
ersi
on
or
enan
tio
mer
ic e
xce
ss (
%)
Fermentation of 3g (in 4L) bicyclo[3.2.0]
hept-2-en-6-one by E. coli B834 pDB5
expressing Rv3049c. (♦ = conversion measured by GC; ■ = enantiomeric excess of (1R, 5S)- (+)-ketone.
Result: 580 mg (19%, >97% e.e.)
single ketone enantiomer
1h
4h
Org. Biomol. Chem. (2006) 4, 1252-1260
Baeyer-Villiger Monooxygenases from M. tuberculosis H37Rv
Bioorg. Med. Chem. Lett. (2006) 16, 4813-4817.
Baeyer-Villiger Monooxygenases from M. tuberculosis H37Rv
E = >200
Bioorg. Med. Chem. Lett. (2006) 16, 4813-4817.
Baeyer-Villiger Monooxygenases from M. tuberculosis H37Rv
‘…the cyclohexanone monooxygenases are particularly overrepresented…’
Proc. Nat. Acad. Sci. USA (2006) 103, 15582-15587
Szolkowy et al. (2009) Chembiochem, in press
Baeyer-Villiger Monooxygenases from R jostii RHA1
Baeyer-Villiger Monooxygenases from R jostii RHA1
Szolkowy et al. (2009) Chembiochem, in press
23 targets
23 PCR and cloned
23 expressed
13 soluble
Baeyer-Villiger Monooxygenases from R jostii RHA1
Szolkowy et al. (2009) Chembiochem, in press
Szolkowy et al. (2009) Chembiochem, in press
Summary 1
• Group I enzymes (MO3, MO4, MO11 and MO20) converted all test substrates and
appeared to be the ‘most active’
• MO14, although a ‘Group II’ enzyme also displayed this wide substrate selectivity
• Other Group II enzymes, MO9 and MO15 did not transform C6 or C7 cyclic ketones
• MO10 and MO18 (Group IV) displayed narrow substrate specificities
• CHMO displayed activities consistent with the literature
Summary 2
• Group I enzymes (MO3, MO4, MO11 and MO20) catalysed poorly enantioselective
transformations of 2-methylcyclopentanone
• MO14, although a ‘Group II’ enzyme was also poorly selective
• Other Group II enzymes, MO9 and MO15 were (R)- selective
• MO18 (Group IV) displayed a respectable E value (25) for this transformation
(-)-9 (-)-10
Summary 3
• Group I enzymes (MO3, MO4, MO11 and MO20) catalyse the formation of the 2-oxa lactone
with poor e.e.
• MO14, also catalyses the exclusive formation of the 2-oxa lactone, but with excellent e.e.
• Other Group II enzymes, MO9 and MO15 catalyse the formation of both 2-oxa and 3-oxa
lactone in good e.e., but derived from the same ketone enantiomer
Szolkowy et al. (2009) Chembiochem, in press
Overall summary
Sequence activity relationships correlated with Groups except MO14
FQRTPN
PGSP
VVVLMICMO9
VVVLMICMO15
LIALMICPAMO
Group II
PIVLMICMO14
PTGGGMVCPMO
PSGGGVTMO20
PSGGGVTMO11
SSATGMVMO4
PTAGGLVMO3
Group I
447674421534469665
Conclusion
• The intragenomic complement of BVMOs in R. jostii RHA1 does indeed reveal a spectrum of
substrate selectivities, regio- and enantioselectivities suggesting that the organism has been
equipped for it job as a ‘catabolic powerhouse’
• In many cases, sequence correlated closely with function – but this is not always true
(see MO14)
• The identification of MO14 as an anomalous enzyme may allow the targeted dissection of the
Determinants of catalytic properties, as illustrated
• Intragenomic compements provide small pool of diversity which may yield extremes of
catalytic behaviour, each acting a sa starting point for directed evolution for catalytic
improvements
Acknowledgements
Funding: CoEBio3, University of Manchester; IRPF UoY, Nuffield Foundation,
ProBio Faraday partnership, Bioscience for Business KTN
Daniel Bonsor
Julianna Solomons
Stephanie Butz
Stephanie Grant
Claudia Szolkowy
Dr Ian Fairlamb
Dr Mark Fogg
Dr Radka Snajdrova
Dr Jared Cartwright
Prof. Lindsay Eltis, UBC, Canada
Prof. Dr. Marko Mihovilovic, TU Vienna
Dr Ian Fairlambwww.biosciencektn.com