Intragenomic Biocatalyst Diversity The family of Baeyer ...ucbepad/RSCBiocatpdf... · Baeyer-Villiger Monooxygenases • Catalyse oxygen insertion adjacent to carbonyl groups Malito

Intragenomic Biocatalyst Diversity

The family of Baeyer-Villiger Monooxygenases from Rhodococcus

jostii RHA1

Gideon Grogan, York Structural Biology Laboratory

[email protected]

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


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


Bioorg. Med. Chem. Lett. (2006) 16, 4813-4817.


E = >200

Bioorg. Med. Chem. Lett. (2006) 16, 4813-4817.


‘…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



23 targets

23 PCR and cloned

23 expressed

13 soluble




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


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

Documents

Intragenomic Biocatalyst Diversity The family of Baeyer ...ucbepad/RSCBiocatpdf... · Baeyer-Villiger Monooxygenases • Catalyse oxygen insertion adjacent to carbonyl groups Malito