BL4101 Enzyme Mechanism

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BL4101Enzyme Mechanism

• James H. Naismith

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Schematic of TB wall

• Unique to mycobacteria (very slow growing)
• resistant to dehydration, immune system etc
• Figure taken from Chem Britain Evan (2000)

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The rhamnose pathway

• dTDP-rhamnose immediate precursor of rhamnose
• Made in four steps
• RmlA glucose-1-phosphate thymidyltransferase (EC
  2.7.7.24)
• RmlB dTDP-D-glucose 4,6-dehydratase (EC4.2.1.46)
• RmlC dTDP-6-deoxy-D-xylo-4-hexulose 3,5-epimerase
  (EC5.1.3.13)
• RmlD dTDP-6-deoxy-L-lyxo-4-hexulose reductase (EC
  1.1.133)

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RmlB
S. Typhimurium solved by mol replacement (Coli
Thoden/Holden)
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dTDP-glucose bound to S. suis (left) S.
typhimurium
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Ligand binding changes structure

• C-terminus orders and covers active site
• Mainchain at active site moves, key residues at
  active site perturbed
• Also seen with dTDP
• Potentiates NAD

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Key contacts

• Triad marked with , Glu 135 and Asp 134
  conserved in all dehydratases
• Tyr 167 acts as principal base (2.6A)
• Thr 133 regulates pKa of O4
• Lys 171 stabilise Tyr 167
• C4 distance TDPG to C4 NAD is 3.2A
• Glu 135 perfectly position to remove H from C5
  position
• O6 strong H-bond to Asp 134 (water bond to 134 in
  xylose)

NADH



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Summary
Role of conserved triad unambiguous, Tyr is
direct base (consistent with recent human GALE
structure SQD1) no proton shuttle Orientation
of NAD and Glucose ring, perfect for hydride
transfer to anti-bonding molecular orbital of
NAD (important check for all substrate
complexes) Asp 134 acts as general acid (now
confirmed) Repulsion between O6 and C6 after bond
cleavage drives sugar into correct orientation
for hydride transfer
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dTDP-xylose bound to S. suis (now 1. 5A)
Water here not O6

• Xylose cannot go beyond first stage (oxidation)
• Ligand appears to be xylose (not a surprise)

• Effectively superimposes with dTDP-glucose
• Density and single xtal spectrum show NADH

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NADH ring bowing important for mechanism
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Class II Aldolases require metals for
function. Fructose-1,6-bisphosphate aldolase
(FBP-aldolase) catalyses the condensation of
dihydroxyacetone phosphate (DHAP) and
glyceraldehyde 3-phosphate (G3P) to produce
fructose 1,6-bisphosphate. This is an aldol
condensation. The enzymes can also catalyse the
reverse reaction namely the cleavage. It is the
latter reaction that occurs in glycolysis. There
are two types of FBP-aldolase called type I and
type II. The type II enzymes are only found in
lower order organisms. Type I use an active site
lysine that covalently attaches to substrate
forming a Schiff base intermediate. The lysine
amine group acts as a nucleophile to attack the
carbonyl group. The Schiff base promotes
formation of an enolate anion by serving as an
electron sink. The enolate anion then adds to
the aldehyde group of G3P and hydrolysis produces
the FBP product. In Class II FBP-aldolases there
is a requirement for a divalent transition metal
cation (Zn 2) AND a monovalent alkali metal
cation (Na or K)
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Reactants and product for Fructose
1,6-bisphosphate aldolase
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The alkali metal is required to form the correct
protein environment to bind DHAP, in particular
the phosphate end of the substrate. The metal
fulfils a structural role and interacts directly
with both the enzyme and the substrate helping to
align the reactive part of the substrate near the
divalent metal ion. We can consider this to be
the binding of an ion pair, DHAP M to the
enzyme. The cation has an octahedral
coordination to 6 oxygens provided by phosphate,
water and main chain carbonyls. The divalent
cation, typically Zn 2 , coordinates to 3
histidines and the carbonyl and hydroxyl oxygens
of DHAP replace solvent. This is a trigonal
bipyramidal geometry. DHAP is aligned for
catalysis as the divalent ion performs a
structural role. The cation also functions as a
Lewis acid and polarises the carbonyl bond of the
ketose substrate. The electrophilic G3P can only
react from the Si face of DHAP. The polarisation
of the carbonyl by the metal increases the
acidity of the hydroxymethylene hydrogens and
supports abstraction of a proton by a glutamic
acid. This generates a carbanion and an
unsaturated CC linkage where addition occurs.
An arginine binds the phosphate of G3P and helps
to bring it into position for the condensation
(formation of a C-C bond). During the reaction
the ene-diolate and the carbonyl acceptor have to
be aligned parallel. An aspartate provides a
proton to convert the carbonyl to a hydroxyl
group as the C-C linkage is formed.
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Step I DHAP and M bind Substrate is aligned
and polarised.
Step II DHAP is deprotonated Carbanion is formed
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Step III G3P binds to the protein and is
aligned with respect to the carbanion
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Step IV C-C bond formation occurs also with
proton transfer from Asp109
Step V FBP leaves the active site
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C-F bond formation in S. cattleya
S. cattleya cell free extract
KF

OHagan et al., Nature 2002 Top 10 chemical
discovery 2002
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5-Fluoro-5-deoxyadenosine synthase

• Specific for F- ion, no detectable turnover with
  other halogens
• Inhibited by SAM analogues such as SAH
• kcat (Deng et al., FEBS 2003) 0.05min-1, Km (SAM)
  0.4mM, Km (F) 8mM
• SN2 displacement ß to oxygen known to be a
  difficult reaction (1920s)

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Monomer structure

• Residue 8 298 ordered
• Two domains, both of which novel (ie no
  structural homologues)
• Large domain 2-180
• Small domain 195-298
• Native structure Rf 20, 1.8Å

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Protein is a hexamer
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Trimer is a key unit

• Extensive contacts
• Protein may have other activity?

• No clues about mechanism from structure
• No function for sequence homologues

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Substrate dragged through purification
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SAM recognition is extensive novel
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Several bidentate hydrogen bonds, anchor substrate
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Ribose very unusual conformation
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Fluorinasegt
Fluorinase
FFPEGTVFATTTYPATGTTTRSVAVRIKQAAKGGARGQWAGSGAGFERAE
114 gi15645332refNP_207503.1
YWPKGSVFVSVVDPGVGTKRKSVVLKTKN---------------------
107 gi15611715refNP_223366.1
YWPKGSVFVSVVDPGVGTNRKSVVLKTKN---------------------
107 gi16801793refNP_472061.1
YWPEATIFVSIVDPGVGSKRRSVVVLTED---------------------
88 gi17228822refNP_485370.1
YFPEGTVHIAVVDPGVGSRRRAIAVEFADG--------------------
94 gi23002712refZP_00046386.1
YWPKGTTFVSVVDPGVGSDRKSIAVKTKS---------------------
89 gi16804625refNP_466110.1
YWPEDTIFVSIVDPGVGSKRRSVAVLTED---------------------
88 gi15902472refNP_358022.1
YWPEGTTFVSVVDPGVGSKRKSVVAKTAK---------------------
97 gi23098522refNP_691988.1
YWPEGTIFISVVDPGVGTDRLSVVAKTKS---------------------
88 gi24380285refNP_722240.1
YWPQGTTFVSVVDPGVGSDRKSVVALTSR---------------------
88 gi23049490refZP_00076624.1
YFPQGTVHIGVVDPGVGTSRRALAIKAGPKGE------------------
90 gi14590374refNP_142440.1
YSPKGTVHVGVIDPGVGTERRAIVIEGDQ---------------------
84 gi21227119refNP_633041.1
YFPAKSVHVGVVDPGVGTSRRALALKAGPEGE------------------
87 gi14521773refNP_127249.1
YSPEGTVHVGVIDPGVGTERRAIVVEGEQ---------------------
84 gi23128187refZP_00110041.1
YFPVGTVHLAVVDPGVGSKRRAIAVEFAQG--------------------
95 gi23041218refZP_00072690.1
YFPSETVHLAVVDPGVGSRRKAIAIQLPNG--------------------
90 gi22299720refNP_682967.1
YFPPETVHIVVVDPGVGTSRRAIALDLEVG--------------------
88 gi15643425refNP_228469.1
DFPPSTVFLVVVDYGVGTSRKAIVMKTKN---------------------
84 gi18314206refNP_560873.1
WFPSGTIFLAVVDPGVGTERLPLIIKT----R------------------
84 gi20093107refNP_619182.1
YFSAKSVHVGVIDPGVGTSRRALAVKAGSKGE------------------
87 gi18976831refNP_578188.1
YSPQGTVHLGVIDPGVGTNRRAIIIEGEQ---------------------
84 gi23024672refZP_00063873.1
YWQPGTVFVSVVDPGVGSKRLSVIAKTTA---------------------
94 gi21674369refNP_662434.1
YFPAETIFVCVVDPGVGTARRAIGVEAGPY--------------------
90 gi15607127refNP_214509.1
YFPEKSVFVGVVDPGVGSERKGIIVKT----E------------------
84 gi20094244refNP_614091.1
WFPPGSVHVGVVDPGVGTERRAVLLEAERG--------------------
86 gi23019435refZP_00059145.1
EFPA-AVHIGVVDPGVGTARRSVALAAGG---------------------
91 gi11498073refNP_069297.1
YFRN-AVHVAVVDPGVGSERRALVIE----GK------------------
82 gi14602006refNP_148551.1
WLPKGSVIVAVVDPGVGTSRYALAVET----E------------------
88 gi15920493refNP_376162.1
YFKKGTIFLVVIDPGVGTERKALLIKT----K------------------
66 gi15669847refNP_248661.1
YFPP-SVHVAVIDPTVGSERKSIVIETKSG--------------------
99 gi23137006refZP_00118717.1
DFPKGSVHLVCVNTPASGKEKLVAIKLEE---------------------
86 gi23473975refZP_00129270.1
YYPAGTLFVAVVDPGVGTGRPLLYAESAG---------------------
86 gi14531032gbAAK63179.1
SFPKGTIHLIGVDIERNKENQHIAMQWND---------------------
87 gi15789578refNP_279402.1
YFPP-AVHLAVVDPGVGTDRSAVVVRAGS---------------------
65 gi22968497refZP_00016084.1
PLPPDSVVIGVVDPGVGGERRAVALRVDG---------------------
125 gi23129872refZP_00111695.1
NPGPSDRLIYHNCAPRQDDPEARRDEGEG---------------------
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Loop crucial for structure of trimer and SAM
binding site
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Mechanism?

• Chemical labelling indicated (OHagan JACS, 2003)
  that C-F formation is SN2 process
• Unusual conformation of ribose ring may increase
  electrophilicity of SAM
• Structure of SAM complex, no water molecule
  nearby and no network of H-bonders that suggested
  a F binding site
• Incubate native protein with KF

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Product bound at same position as SAM

• Refined Rf 24 to 2.7Å, isomorphous with
  native ring torsion unclear

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Fluoride binding pocket

• Strong H-bond from F to amide backbone
• Distance F - N 3.1Å
• Second polar contact to Ser (cf glycosidase)
• Distance F O 3.5 Å
• Otherwise hydrophobic
• Pocket with radius 1.4-1.6 Å
• Cl-, Br- all too big

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Mechanism
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SN2 displacement

• Orientation minimises leaving and attacking group
  interaction with oxygen lone pair
• Ring torsion prevents anomeric involvement of
  ring oxygen with adenine and no H-bonds to ring
  oxygen (would hinder reaction by charging oxygen)
• Ring strain on C5 position

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Chlorination

• Chlorinated brominated natural products are
  very common gt 2000 known
• Important class of molecules includes many
  antibiotics, eg vancomycin, rebeccamycin
• Haloperoxidase mechanism using iron / vanadium
  based chemistry assumed to used in bacteria
  despite problems
• Perhydrolases are not important in halogenation
• Gene sequencing showed halo peroxidase assumption
  wrong

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Chlorinated natural products
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The Walsh mechanism
Yeh et al., 2005 PNAS
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Reaction

• 3 chlorinases isolated and characterised from
  Pseudomonas species (65kDa)

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Comes from pyrrolnitrin pathway
Potent antifungacide made by Pseudomonas
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7-chlorotrytophan synthase (PrnA)

• FAD module pink, substrate module cyan
• Only one Cl- ion bound close to FAD
• Cl-Trp and Trp identical
• FAD and Trp over 10Å apart

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Protein is a dimer

• FADH2 leads to disorder in loops at Trp binding
  site.
• A loop at flavin binding site is disordered upon
  Trp binding.
• This suggests possible communication between the
  sites.

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Experimental density
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Mechanism

• Walsh and Van Pee mechanisms must be incorrect
• FAD and Trp (Cl-Trp) too far apart for direct
  transformation
• No room at the FAD for substrate
• No evidence for the massive conformation change
  required to either allow Trp to bind at FAD or to
  allow Trp to move to FAD (7 structures)

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Flavin monooxygenase (PHBH) similar topology to
FAD module of PrnA

• Only FAD module superimposes as predicted
• In PHBH there is room at the FAD for binding of
  substrate.
• The structure shows this is impossible in PrnA.
  No room, in addition two conserved Trps in PrnA
  mask FAD from solvent

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Mechanism of monooxygenase (PHBH)
Work of Massey, Ballou Biochemistry. 2001, 37,
11156-67 (others)
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By analogy, halogenase generates HOCl

• Attack of Cl- is at the most electropositive
  oxygen (cf PHBH) and HOCl is produced by peroxide
  and Cl-. (The other oxygen would be expected to
  be more electron rich. Peroxychloride is highly
  reactive forming HOCl and water.)
• Flavin peroxide is much more reactive than free
  peroxide to nucleophilic attack.
• General mechanism for all halogenases, since FAD
  domain conserved

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Chemical evidence for HOCl

• MCD is a highly reactive tracer for free HOCl.
• None detected
• Due to presence of NADH in assay, cannot rule
  small amounts but any free HOCl is being
  destroyed by NADH faster than it reacts with MCD
  (the most reactive trap for HOCl)
• 5-MI only slowly reacts with free HOCl
• Reaction occurs at enzyme
• Reaction identical to reaction with free HOCl
• Inhibits PrnA

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Problems

• HOCl is not reactive enough
• HOCl although reactive will not chlorinate
  tryptophan (or several other aromatics) in
  solution
• How is regioselectivity achieved?
• HOCl is not selective

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Unreactive channel connects active sites
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Mechanism

• Lys by donating a proton, makes Cl much more
  electropositive (reactive to nucleophilc attack)
• Glu stabilises Wheland complex and re-aromatises
  substrate
• Alternative formation of chloramine