Predicting

Protein

Function

from

Structure

The

Roles

of

Short-chain

Dehydrogenase/Reductase

Enzymes

in

Bordetella

O-antigen

Biosynthesis

The

pathogenic

bacteria

Bordetella

parapertussis

and

Bordetella

bronchiseptica

express

a

lipopolysaccharide

O

antigen

containing

a

polymer

of

acid

The

O-antigen

cluster

contains

three

neighbouring

genes

that

encode

proteins

belonging

to

the

short-chain

dehydrogenase/reductase

-LRB-

SDR

-RRB-

family

wbmF

wbmG

and

wbmH

and

we

aimed

to

elucidate

their

individual

functions

Mutation

and

complementation

implicate

each

gene

in

O-antigen

expression

but

as

their

putative

sugar

nucleotide

substrates

are

not

currently

available

biochemical

characterisation

of

WbmF

WbmG

and

WbmH

is

impractical

at

the

present

time

SDR

family

members

catalyse

a

wide

range

of

chemical

reactions

including

oxidation

reduction

and

epimerisation

Because

they

typically

share

low

sequence

conservation

however

catalytic

function

can

not

be

predicted

from

sequence

analysis

alone

In

this

context

structural

characterisation

of

the

native

proteins

co-crystals

and

small-molecule

soaks

enables

differentiation

of

the

functions

of

WbmF

WbmG

and

WbmH

These

proteins

exhibit

typical

SDR

architecture

and

coordinate

NAD

In

the

substrate-binding

domain

all

three

enzymes

bind

uridyl

nucleotides

WbmG

contains

a

typical

SDR

catalytic

TYK

triad

which

is

required

for

oxidoreductase

function

but

the

active

site

is

devoid

of

additional

acid

base

functionality

Similarly

WbmH

possesses

a

TYK

triad

but

an

otherwise

feature-poor

active

site

Consequently

function

can

probably

be

ruled

out

for

these

enzymes

The

WbmF

active

site

contains

conserved

features

namely

a

positionally

conserved

cysteine

-LRB-

-RRB-

and

basic

side

chain

-LRB-

or

-RRB-

but

lacks

the

serine/threonine

component

of

the

SDR

triad

and

therefore

may

not

act

as

an

oxidoreductase

The

data

suggest

a

pathway

for

synthesis

of

the

O-antigen

precursor

UDP-2,3-diacetamido-2,3-dideoxy-l-galacturonic

acid

and

illustrate

the

usefulness

of

structural

data

in

predicting

protein

function

Introduction

The

acceleration

of

genome

sequencing

has

created

a

challenge

for

modern

biology

namely

interpretation

of

the

vast

and

growing

amount

of

genetic

information

in

the

databases

Search

algorithms

such

as

the

basic

local

alignment

search

tool

-LRB-

BLAST

-RRB-

can

efficiently

find

related

genes

but

only

in

a

small

minority

of

cases

is

the

precise

function

of

a

novel

enzyme

equivalent

to

that

of

the

highest

scoring

hit

In

straightforward

cases

the

output

is

useful

to

generate

hypotheses

for

the

novel

enzyme

's

function

based

on

common

catalytic

chemistry

substrates

or

both

and

these

postulates

are

then

amenable

to

direct

biochemical

investigation

To

make

full

use

of

genome

data

it

is

important

that

where

such

standard

bioinformatic

analysis

can

not

predict

precise

roles

and

biochemical

function

assays

are

not

possible

tools

are

developed

to

differentiate

the

functions

of

genes

A

good

example

of

such

a

challenging

case

is

where

gene

products

are

annotated

as

short-chain

dehydrogenase/reductases

-LRB-

SDRs

-RRB-

Members

of

this

family

share

low

sequence

identity

-LRB-

typically

-RRB-

and

catalyse

a

wide

range

of

chemical

reactions

including

oxidation

reduction

epimerisation

dehydration

and

decarboxylation

-LRB-

reviewed

in

Ref

-RRB-

Bordetella

parapertussis

and

Bordetella

bronchiseptica

are

pathogens

of

mammals

B.

bronchiseptica

is

associated

with

respiratory

tract

infections

in

many

animals

including

acute

tracheobronchitis

in

dogs

-LRB-

kennel

cough

-RRB-

and

atrophic

rhinitis

in

pigs

whereas

B.

parapertussis

can

cause

whooping

cough

in

humans

A

separate

lineage

of

B.

parapertussis

infects

sheep

The

major

component

of

the

outer

leaflet

of

the

outer

membrane

in

Gram-negative

bacteria

is

lipopolysaccharide

-LRB-

LPS

-RRB-

The

complete

structure

of

this

macromolecule

in

B.

bronchiseptica

and

B.

parapertussis

has

been

recently

described

The

lipid

A

domain

of

LPS

which

consists

of

a

diglucosamine

backbone

substituted

with

fatty

acyl

chains

forms

the

outer

leaflet

of

the

outer

membranes

of

the

bacteria

Lipid

A

is

linked

to

a

complex

branching

oligosaccharide

known

as

the

core

Lipid

A-core

comprises

a

proportion

of

the

LPS

that

is

exposed

on

the

cell

surface

and

is

known

as

band

B

In

B.

bronchiseptica

lipid

A-core

can

be

substituted

with

a

trisaccharide

and

this

structure

is

known

as

band

A.

Synthesis

of

the

band

A

trisaccharide

requires

functions

encoded

in

the

wlb

gene

cluster

-LRB-

previously

named

bpl

-RRB-

Both

B.

bronchiseptica

and

B.

parapertussis

also

substitute

their

LPS

with

an

O

antigen

This

O

antigen

contains

to

2,3-diacetamido-2,3-dideoxy-l-galactosaminuronic

acid

-LRB-

-RRB-

and

is

required

for

full

virulence

in

animal

and

in

vitro

models

of

infection

O-antigen

biosynthesis

requires

genes

in

the

wbm

cluster

that

is

adjacent

to

the

wlb

genetic

locus

The

wbm

locus

contains

three

neighbouring

SDR

genes

-LRB-

wbmF

wbmG

and

wbmH

-RRB-

all

of

which

have

been

annotated

as

nucleotide-sugar

epimerases/dehydratases

The

structure

of

complete

O

antigen

and

the

homology

of

wbm

genes

with

genes

of

known

function

have

led

us

to

propose

a

pathway

for

biosynthesis

of

this

polysaccharide

and

we

are

currently

testing

various

steps

in

this

scheme

as

part

of

an

ongoing

project

to

determine

the

functions

of

all

wbm

genes

This

report

specifically

concerns

the

roles

of

the

SDR

genes

wbmF

wbmG

and

wbmH

O-antigen

residues

are

synthesised

as

sugar

nucleotide

precursors

The

probable

substrate

for

wbm

locus-encoded

biosynthesis

of

the

nucleotide-activated

is

UDP-2,3-diacetamido-2,3-dideoxy-d-mannuronic

acid

-LRB-

-RRB-

This

compound

is

related

to

the

l-galacto

configuration

by

inversion

of

the

stereochemistry

at

the

hexose

and

positions

In

the

bordetellae

is

formed

as

a

precursor

for

band

A

trisaccharide

synthesis

by

of

UDP-2,3-diacetamido-2,3-dideoxy-d-glucuronic

acid

catalysed

by

WlbD

Because

there

are

precedents

for

either

a

or

SDR

enzymes

catalysing

conversions

of

sugar

nucleotides

we

hypothesise

that

one

or

more

of

WbmF

WbmG

and

WbmH

operate

in

this

biosynthetic

pathway

to

catalyse

the

conversion

of

to

acid

-LRB-

-RRB-

Because

of

the

low

percentage

conversion

of

the

WlbD-catalysed

reaction

is

not

currently

available

for

biochemical

studies

and

therefore

this

hypothesis

can

not

be

directly

tested

at

the

present

time

In

this

report

we

demonstrate

the

involvement

of

wbmF

wbmG

and

wbmH

in

O-antigen

expression

by

mutation

of

the

genes

in

B.

bronchiseptica

Because

the

putative

substrate

is

unavailable

we

elected

to

probe

the

catalytic

functions

of

WbmF

WbmG

and

WbmH

by

characterising

these

proteins

three-dimensional

structures

With

soaking

and

co-crystallisation

experiments

densities

corresponding

to

cofactors

and

nucleotide

portions

of

substrate

analogues

can

be

identified

within

the

active

sites

Biochemical

and

crystallographic

studies

have

defined

the

structural

basis

for

the

catalytic

chemistries

of

a

range

of

other

SDR

enzymes

-LRB-

reviewed

in

Ref

-RRB-

Our

knowledge

of

these

structure

function

relationships

enables

us

to

interpret

the

structures

of

the

wbm

SDRs

We

have

analysed

the

potential

for

acid

base

chemistry

in

the

active

sites

to

differentiate

their

potential

roles

in

vivo

Based

on

the

composition

of

O

antigen

the

genetic

context

of

these

wbm

genes

and

information

from

the

structural

studies

we

propose

roles

for

these

genes

in

O-antigen

biosynthesis

The

results

demonstrate

the

usefulness

and

limitations

of

X-ray

data

in

elucidating

biochemical

pathways

where

catalytic

activity

can

not

be

directly

measured

Results

Mutational

analysis

of

wbmF

wbmG

and

wbmH

To

test

the

hypothesis

that

wbmF

wbmG

and

wbmH

are

involved

in

O-chain

biosynthesis

each

gene

was

disrupted

by

insertion

of

a

tetracycline-resistance

cassette

Mutation

of

the

chromosomal

genes

was

confirmed

by

Southern

hybridisation

-LRB-

data

not

shown

-RRB-

The

effects

of

these

mutations

were

assessed

by

silver-stained

SDS-PAGE

analysis

and

immunoblotting

of

mutant

LPS

-LRB-

Fig.

-RRB-

Mutation

of

wbmG

or

wbmH

results

in

apparent

abrogation

of

O-antigen

synthesis

as

the

LPS

from

these

mutants

lacks

O

band

as

detected

by

either

silver

stain

or

Western

blot

O-antigen

expression

in

the

wbmF

mutant

is

dramatically

reduced

compared

with

the

wild

type

but

this

strain

retains

its

ability

to

express

a

small

amount

of

material

with

the

electrophoretic

mobility

of

O-band

LPS

and

which

binds

the

O-antigen-specific

monoclonal

antibody

Each

of

the

mutations

was

complemented

by

expression

of

the

cognate

wild-type

allele

in

trans

-LRB-

Fig.

-RRB-

Each

coding

sequence

was

cloned

into

the

broad

host

range

vector

-LRB-

kan

-RRB-

under

the

control

of

the

flaA

promoter

to

generate

complementation

vectors

Complementation

of

the

wbmG

and

wbmH

mutations

restored

O-antigen

expression

to

near

wild-type

levels

O-antigen

expression

in

the

complemented

wbmF

mutant

was

increased

relative

to

the

wbmF

mutant

but

was

still

considerably

less

than

in

wild

type

The

reason

for

incomplete

complementation

of

the

wbmF

mutation

is

unknown

but

is

not

likely

to

be

due

to

polar

effects

of

the

mutation

because

due

to

the

organisation

of

genes

in

the

wbm

locus

wbmF

is

at

the

end

of

an

operon

Introduction

of

the

empty

complementation

vector

had

no

effect

on

O-antigen

expression

in

any

of

the

mutants

The

gene

products

WbmF

WbmG

and

WbmH

are

all

in

the

extended

SDR

family

The

SDR

family

is

divided

into

the

classical

and

extended

subfamilies

primarily

on

the

basis

of

protein

length

and

the

sequence

pattern

of

the

glycine-rich

cofactor-binding

region

close

to

the

N

terminus

According

to

both

criteria

the

products

of

wbmF

wbmG

and

wbmH

which

are

predicted

to

have

and

amino

acids

respectively

and

all

have

the

GxxGxxG

motif

near

the

N

terminus

are

extended

-LRB-

Fig.

-RRB-

WbmG

and

WbmH

are

closely

related

to

each

other

sharing

amino

acid

identity

WbmF

WbmG

and

WbmH

are

all

homologous

to

-LRB-

NAD-dependent

epimerase/dehydratase

family

-RRB-

The

most

closely

related

characterised

homologues

of

these

Wbm

SDRs

in

the

protein

databases

are

deoxythymidine

diphosphate

-LRB-

dTDP

-RRB-

glucose

from

Escherichia

coli

and

Streptomyces

venezuelae

with

or

sequence

identity

-LRB-

Table

-RRB-

This

level

of

sequence

conservation

between

members

of

the

SDR

family

does

not

imply

conservation

of

either

catalytic

chemistry

or

substrate

rather

this

functional

information

can

be

better

determined

by

examination

of

the

residues

at

key

positions

in

the

active

sites

and

binding

pockets

Several

conserved

motifs

within

the

extended

SDR

subfamily

facilitate

alignment

of

sections

of

the

Wbm

SDR

sequences

with

homologues

-LRB-

Fig.

-RRB-

but

many

of

the

critical

functional

amino

acids

are

located

outside

of

these

motifs

and

with

this

low

level

of

conservation

it

is

not

possible

to

make

full

sequence

alignments

with

confidence

that

all

of

these

residues

are

correctly

identified

Crystallisation

of

and

Thus

far

the

data

implicated

each

of

these

wbm

SDRs

in

O-antigen

synthesis

but

did

not

indicate

precise

roles

for

each

gene

Therefore

in

order

to

elucidate

their

individual

functions

we

characterised

the

gene

products

by

X-ray

crystallography

The

overexpression

purification

and

crystallisation

of

these

proteins

are

described

elsewhere

The

structures

discussed

in

the

present

work

are

summarised

in

Table

General

architecture

of

and

These

proteins

all

exhibit

the

typical

SDR

family

architecture

-LRB-

Fig.

-RRB-

The

structures

each

comprise

two

domains

The

first

of

these

is

the

N-terminal

Rossmann

fold

domain

in

which

a

central

β

sheet

is

flanked

by

two

layers

of

α

helices

The

cofactor-binding

motif

GxxGxxG

is

located

at

the

C-terminal

edge

of

this

β

sheet

which

has

seven

parallel

β

strands

running

in

the

order

The

second

domain

is

largely

made

up

of

C-terminal

sequence

and

contains

all

of

the

residues

involved

in

binding

of

the

nucleotide

portion

of

the

substrate

The

catalytic

sites

in

SDR

enzymes

are

located

at

the

interface

of

these

two

domains

where

in

sugar-nucleotide-modifying

enzymes

the

substrate

hexose

is

brought

into

proximity

with

the

cofactor

nicotinamide

ring

The

main

architectural

differences

between

these

three

proteins

lie

in

their

C-terminal

regions

-LRB-

Fig.

-RRB-

The

C

terminus

of

consists

of

a

loop

that

stretches

out

from

the

C-terminal

domain

to

interact

with

the

Rossmann

domain

In

this

loop

is

not

visible

in

the

density

although

this

difference

may

not

reflect

any

distinction

between

the

real

structures

of

the

two

proteins

in

solution

In

WbmF

the

last

residues

form

a

large

bent

helix

that

covers

two

faces

of

the

C-terminal

domain

There

is

also

an

insertion

of

amino

acids

in

WbmF

relative

to

the

other

structures

including

all

residues

from

to

This

extra

loop

extends

over

the

nicotinamide

end

of

the

cofactor-binding

pocket

SDRs

typically

form

dimers

or

higher

oligomers

The

crystal

packing

of

these

proteins

suggests

that

WbmF

WbmG

and

WbmH

have

the

conserved

four-helix

bundle

usually

found

at

the

dimer

interface

Analytical

gel

filtration

chromatography

indicated

a

hydrodynamic

radius

for

each

of

these

proteins

consistent

with

either

a

monomer

or

dimer

-LRB-

data

not

shown

-RRB-

We

conclude

therefore

that

these

proteins

are

present

in

solution

predominantly

in

dimer

form

In

the

WbmG

and

WbmH

structures

the

dimer

interaction

buries

Å2

-LRB-

of

total

protein

surface

-RRB-

and

Å2

-LRB-

-RRB-

of

surface

per

dimer

respectively

In

the

WbmF

structure

the

interface

region

is

extended

to

include

a

short

antiparallel

β

sheet

composed

of

the

residues

to

from

each

monomer

as

well

as

the

loops

that

connect

these

strands

to

the

bundle

-LRB-

to

-RRB-

Å2

is

buried

in

the

WbmF

dimer

interface

which

is

equivalent

to

of

the

total

protein

surface

In

all

three

cases

the

majority

of

the

residues

buried

at

these

interfaces

are

hydrophobic

or

aromatic

Cofactor

binding

NAD

can

be

modelled

into

the

electron

density

in

all

three

proteins

and

copurify

with

this

compound

from

the

E.

coli

expression

host

but

in

order

to

stabilise

the

protein

in

solution

required

the

addition

of

exogenous

cofactor

and

this

is

the

most

likely

source

of

this

molecule

in

these

structures

The

preference

in

each

case

for

NAD

cofactor

as

opposed

to

NADP

is

indicated

by

the

close

interaction

-LRB-

Å

hydrogen

bonds

-RRB-

of

an

aspartate

side

chain

with

the

hydroxyl

of

the

adenylic

ribose

This

side

chain

would

disfavour

binding

of

NADP

by

both

steric

and

electrostatic

repulsion

of

the

NADP

phosphate

In

some

SDR

structures

-LSB-

e.g.

reductase

-LRB-

GMER

-RRB-

and

-RSB-

the

bound

cofactor

has

direct

access

to

solvent

indicating

a

pathway

via

which

spent

NAD/NADP

can

be

replaced

from

solution

In

these

structures

the

bound

cofactor

is

buried

within

the

core

of

the

protein

in

the

case

of

the

structure

the

insertion

supplies

an

additional

occluding

loop

further

blocking

release

of

cofactor

in

this

conformation

Binding

of

substrate

Soaking

and

co-crystallisation

experiments

with

and

revealed

the

binding

mode

for

the

nucleotide

portion

of

the

sugar

nucleotide

substrates

of

these

enzymes

-LRB-

Fig.

-RRB-

The

X-ray

data

from

a

crystal

soaked

with

UDP-glucose

revealed

extra

density

that

could

be

modelled

as

far

as

the

α-phosphate

The

location

of

this

molecule

is

consistent

with

the

nucleotide-binding

site

for

SDR

homologue

structures

and

with

the

hydrogen-bonding

chemistry

expected

to

bind

uridine

No

density

was

visible

corresponding

to

the

hexose

portion

of

the

soaked

compound

nor

the

second

phosphate

suggesting

that

either

this

portion

of

the

molecule

is

disordered

within

the

crystal

or

the

compound

was

partially

hydrolysed

and

the

smaller

uridine

monophosphate

-LRB-

UMP

-RRB-

preferentially

soaked

into

the

substrate-binding

pocket

In

either

case

the

data

indicate

that

the

moiety

of

UDP-glucose

is

not

strongly

bound

in

a

single

conformation

by

WbmG

A

similar

soaking

experiment

but

with

GDP-mannose

did

not

indicate

any

binding

of

this

sugar

nucleotide

-LRB-

data

not

shown

-RRB-

indicating

a

possible

preference

for

the

uracil

base

The

hydrogen

bonding

between

the

WbmG

pocket

and

UMP

suggests

a

basis

for

this

preference

-LRB-

Fig.

-RRB-

Although

we

were

unable

to

obtain

any

complex

of

WbmH

with

a

substrate

analogue

bound

the

nucleotide-binding

domain

of

WbmH

is

conserved

in

sequence

and

structure

with

WbmG

implying

identical

specificity

and

a

common

binding

mode

for

the

nucleoside

portion

of

their

substrates

-LRB-

Fig.

-RRB-

UDP

density

to

the

α-phosphate

is

also

visible

in

the

WbmF/NAD/UDP

ternary

complex

The

UMP

density

in

this

structure

and

the

chemistry

of

its

interaction

with

the

protein

suggests

that

this

protein

like

WbmG

and

WbmH

recognises

a

UDP-sugar

substrate

Both

α

and

β

nucleotide

phosphates

could

be

modelled

in

the

data

from

a

UDP

soak

into

a

WbmF-NAD

co-crystal

and

co-crystallisation

of

with

UDP

In

both

structures

the

β-phosphate

is

coordinated

by

a

conserved

arginine

-LRB-

Fig.

and

b

-RRB-

although

the

precise

location

of

this

phosphate

is

different

in

the

two

structures

and

both

are

inconsistent

with

the

β-phosphate

positions

of

bound

substrate

in

the

structure

of

a

mutant

of

dTDP-glucose

DesIV

from

S.

venezuelae

-LRB-

Fig.

-RRB-

In

this

DesIV

structure

-LSB-

Protein

Data

Bank

-LRB-

PDB

-RRB-

ID

-RSB-

the

position

of

the

substrate

is

consistent

with

the

necessary

overlap

of

molecular

orbitals

required

for

hydride

transfer

from

the

glucose

to

the

cofactor

It

is

likely

therefore

that

the

β-phosphate

positions

in

WbmF

and

WbmG

structures

do

not

represent

the

positions

of

equivalent

phosphates

when

the

native

substrates

for

these

enzymes

are

bound

in

the

active

site

One

common

feature

of

substrate

binding

in

SDR

enzymes

is

pi

stacking

of

the

nucleobase

with

an

amino

acid

side

chain

In

WbmF

this

interaction

involves

-LRB-

Fig.

-RRB-

Unusually

in

WbmG

the

pi-stacking

residue

-LRB-

-RRB-

is

the

one

that

hydrogen

bonds

with

the

uracil

-LRB-

Fig.

-RRB-

In

WbmF

E.

coli

UDP-galactose

-LRB-

GalE

-RRB-

S.

venezuelae

DesIV

and

Pseudomonas

aeruginosa

WbpP

the

hydrophobic

contact

is

provided

by

the

residue

in

the

n

position

whose

backbone

amide

hydrogen

bonds

with

the

base

's

group

-LRB-

Fig.

-RRB-

This

unusual

feature

of

the

substrate

binding

in

WbmG

could

not

have

been

predicted

from

sequence

alignments

alone

Potential

catalytic

chemistry

SDR

enzymes

have

a

conserved

catalytic

triad

that

is

involved

in

their

oxidoreductase

activity

and

that

is

classically

composed

of

a

spatially

conserved

serine

tyrosine

and

lysine

-LRB-

SYK

-RRB-

triad

for

example

and

in

WbpP

The

first

member

of

this

triad

is

sometimes

found

as

a

threonine

-LRB-

TYK

-RRB-

and

the

tyrosine

can

be

replaced

by

a

methionine

-LRB-

SMK

-RRB-

The

SDR

triad

is

conserved

in

WbmG

and

WbmH

as

TYK

but

in

WbmF

superimposes

onto

the

Ser/Thr

position

-LRB-

Fig.

-RRB-

In

the

context

of

O-antigen

synthesis

in

Bordetella

we

are

interested

in

the

potential

of

the

wbm

SDR

enzymes

to

catalyse

secondary

reactions

of

their

substrates

beyond

the

oxidoreductase

reactions

common

to

all

SDRs

Of

particular

significance

is

the

identification

of

a

potential

Despite

the

lack

of

a

structure

in

which

a

full

sugar

nucleotide

substrate

analogue

is

bound

the

cavity

into

which

the

substrate

hexose

binds

is

easily

identifiable

by

comparison

with

homologue

structures

and

because

it

is

necessarily

interposed

between

the

UMP-binding

site

and

the

cofactor

nicotinamide

ring

The

active

sites

of

WbmG

and

WbmH

are

devoid

of

amino

acid

side

chains

capable

of

acid

base

chemistry

except

for

their

respective

TYK

triads

and

in

WbmH

SDR

enzymes

that

catalyse

of

their

substrates

have

a

spatially

conserved

catalytic

cysteine

and

a

basic

side

chain

In

GMER

these

residues

are

and

in

GDP-mannose

-LRB-

GME

-RRB-

they

are

and

In

WbmG

and

WbmH

these

amino

acids

superimpose

onto

hydrophobic

side

chains

but

in

WbmF

this

catalytic

cysteine

is

conserved

-LRB-

-RRB-

and

there

are

two

candidates

for

the

basic

side

chain

and

-LRB-

Fig.

-RRB-

is

disordered

in

all

of

our

WbmF

crystals

indicating

that

an

element

of

induced

fit

may

be

required

to

bring

all

of

the

active-site

components

into

place

has

a

very

unusual

backbone

conformation

with

a

cis

peptide

bond

to

the

following

residue

This

is

strongly

suggestive

of

a

functional

role

for

this

residue

since

such

conformations

are

rarely

observed

except

where

they

are

required

to

place

a

key

side

chain

in

the

active

site

Modelling

of

the

putative

substrate

into

the

WbmF

active

site

The

analysis

of

the

potential

catalytic

chemistry

of

WbmF

suggested

that

its

role

in

O-antigen

biosynthesis

may

be

to

catalyse

the

required

in

the

overall

conversion

of

to

The

derivative

of

is

therefore

a

likely

substrate

or

reaction

intermediate

for

WbmF

This

compound

contains

a

bulkier

sugar

than

any

of

the

substrates

of

the

characterised

sugar-nucleotide-modifying

SDRs

An

important

test

of

our

proposed

pathway

is

that

the

enzymes

must

be

able

to

accommodate

these

unusually

large

substrates

We

modelled

into

the

active

site

of

WbmF

-LRB-

Fig.

-RRB-

This

showed

that

it

is

feasible

for

the

substrate

to

occupy

the

active

site

of

WbmF

in

a

manner

that

is

consistent

with

the

experimentally

determined

binding

site

for

UMP

the

proximity

of

the

most

likely

catalytic

site

and

good

geometry

Discussion

The

abrogation

or

reduction

of

O-antigen

expression

as

a

result

of

wbmF

wbmG

or

wbmH

mutations

implicates

all

three

genes

in

the

biosynthesis

of

this

molecule

and

is

consistent

with

the

hypothesis

that

these

three

adjacent

SDR

genes

catalyse

the

to

conversion

which

is

probably

required

for

O-antigen

synthesis

Structural

analysis

of

these

three

wbm

SDR

gene

products

also

enables

us

to

distinguish

between

them

in

terms

of

potential

catalytic

chemistry

Both

WbmG

and

WbmH

have

the

conserved

SDR

catalytic

triad

-LRB-

TYK

in

these

cases

-RRB-

which

suggests

that

they

should

be

competent

to

function

as

oxidoreductases

In

both

of

these

enzymes

the

pocket

that

surrounds

the

active

site

is

hydrophobic

and

featureless

compared

with

other

SDR

enzymes

the

lack

of

side

chains

capable

of

acid

base

chemistry

indicates

that

the

functions

of

these

enzymes

are

likely

to

be

limited

to

oxidation

and/or

reduction

and

probably

rules

out

specific

activity

The

hydroxyl

of

in

WbmH

may

be

capable

of

facilitating

proton

exchange

however

an

amino

acid

pair

capable

of

acid

base

chemistry

is

required

for

function

Some

SDRs

that

catalyse

both

oxidation

and

reduction

bind

cofactor

irreversibly

and

recycle

it

within

the

active

site

In

other

SDRs

this

binding

is

reversible

and

the

cofactor-binding

site

is

situated

at

the

bottom

of

a

solvent-accessible

groove

-LRB-

e.g.

in

E.

coli

GMER

PDB

ID

-RRB-

No

such

groove

is

visible

in

the

WbmF

WbmG

and

WbmH

structures

where

the

cofactor-binding

sites

are

almost

completely

occluded

However

we

do

not

take

this

observation

as

being

diagnostic

of

irreversible

cofactor

binding

in

these

proteins

for

the

following

reasons

Firstly

exogenous

cofactor

is

required

for

WbmF

to

be

stable

in

solution

with

respect

to

precipitation

Presumably

either

cofactor

that

copurifies

with

WbmF

can

be

released

resulting

in

unfolding

and

aggregation

of

the

protein

or

apo-WbmF

must

bind

cofactor

supplied

from

the

medium

in

order

to

fold

correctly

Either

case

would

suggest

that

NAD

can

be

exchanged

between

WbmF

and

solvent

Secondly

human

GalE

reversibly

binds

cofactor

and

yet

does

not

have

an

open

binding

site

like

that

of

GMER

Finally

a

conversion

of

configuration

from

d-manno

to

l-galacto

can

be

catalysed

by

a

single

tight

NAD

binding

enzyme

such

as

GME

but

we

can

not

envisage

a

pathway

in

which

two

or

three

such

enzymes

contribute

to

this

transformation

Instead

we

suggest

that

the

protein

loops

that

cover

the

NAD-binding

site

may

be

flexible

and

move

to

allow

release

and

replacement

of

spent

cofactor

WbmF

lacks

the

serine/threonine

of

the

SDR

catalytic

triad

The

conservation

of

the

classical

SDR

catalytic

triad

is

not

absolute

amongst

members

of

the

family

For

example

in

the

very

unusual

SDR

enzyme

biliverdin

IXβ

reductase

-LRB-

which

catalyses

the

reduction

of

a

range

of

substrates

including

some

flavins

and

non-α-isomers

of

biliverdin

-RRB-

a

histidine

occupies

the

usual

place

of

the

catalytic

lysine

and

tyrosine

residues

The

shapes

and

electron

delocalisation

exhibited

by

biliverdin

IXβ

reductase

substrates

imply

that

compared

with

the

putative

substrates

of

the

Wbm

SDR

enzymes

they

will

have

rather

special

requirements

for

binding

geometry

and

reaction

intermediate

stabilisation

during

catalysis

In

those

SDRs

that

oxidise

or

reduce

sugar

nuclotides

at

the

hexose

position

however

the

catalytic

triad

serine/threonine

residue

is

important

because

it

modifies

the

pKa

of

the

hydroxyl

by

forming

a

short

hydrogen

bond

Mutation

of

the

triad

threonine

-LRB-

-RRB-

to

alanine

in

E.

coli

dTDP-Glc

results

in

a

drop

in

kcat

whereas

mutation

of

to

alanine

in

E.

coli

GalE

reduces

epimerase

activity

-LRB-

kcat

-RRB-

almost

The

incomplete

WbmF

catalytic

triad

raises

doubts

about

the

competence

of

this

enzyme

as

a

sugar

nucleotide

oxidoreductase

It

is

possible

that

the

function

of

the

missing

side

chain

is

provided

by

another

active-site

residue

this

implies

a

different

substrate-binding

geometry

and

further

studies

will

be

required

to

investigate

this

possibility

Conversely

if

a

standard

substrate-binding

orientation

is

assumed

the

active

site

of

WbmF

resembles

that

of

and

our

structures

of

this

protein

identify

and

either

or

as

potential

mediators

of

proton

exchange

in

the

epimerisation

reaction

one

group

abstracts

a

proton

from

the

sugar

while

the

other

donates

a

proton

to

the

opposite

face

By

modelling

the

compound

into

the

WbmF

structure

we

have

demonstrated

that

there

is

space

within

this

active

site

to

accommodate

this

bulky

sugar

nucleotide

Other

solutions

produced

in

the

modelling

process

demonstrated

alternative

plausible

substrate

positions

-LRB-

data

not

shown

-RRB-

For

this

reason

we

do

not

suggest

that

the

solution

presented

here

-LRB-

Fig.

-RRB-

represents

the

true

active

conformation

While

we

did

not

attempt

similar

modelling

experiments

with

WbmG

and

WbmH

visual

inspection

of

these

structures

indicates

that

they

also

have

spacious

active

sites

compared

with

other

SDRs

and

can

therefore

accommodate

diacetamido

uronic

acids

in

their

hexose-binding

pockets

We

therefore

propose

the

following

biosynthetic

pathway

-LRB-

Fig.

-RRB-

for

the

of

One

of

the

putative

oxidoreductases

WbmG

or

WbmH

catalyses

the

initial

oxidation

at

and

the

other

oxidoreductase

is

responsible

for

the

final

reduction

step

Introduction

of

the

keto

group

lowers

the

pKa

of

the

and

hydrogen

atoms

allowing

WbmF

to

catalyse

proton

exchange

effecting

This

scheme

is

the

most

consistent

with

all

of

the

available

data

The

function

suggested

for

WbmF

is

very

unusual

in

that

we

suggest

that

this

enzyme

does

not

catalyse

oxidation

or

reduction

of

its

substrate

If

this

is

the

case

WbmF

would

be

the

first

such

member

of

the

SDR

family

and

this

would

imply

that

the

cofactor

in

WbmF

has

a

purely

structural

role

without

participating

in

the

catalytic

cycle

It

has

been

suggested

that

the

transcriptional

regulation

protein

NmrA

from

Aspergillus

nidulans

is

a

NAD-binding

SDR

family

member

that

is

not

an

enzyme

at

all

NmrA

appears

to

function

in

control

of

nitrogen

metabolism

through

physical

interaction

with

the

GATA

family

transcription

factor

AreA

possibly

by

controlling

the

rate

of

entry

of

AreA

into

the

nucleus

and

the

structural

equivalents

of

the

SDR

triad

tyrosine

and

serine/threonine

in

NmrA

are

and

respectively

Since

the

NmrA

structure

was

reported

however

several

SDR

enzymes

with

SMK

triads

have

been

reported

-LRB-

e.g.

WbpM

from

P.

aeruginosa

-RRB-

so

that

the

nonconservation

of

the

SDR

catalytic

triad

in

NmrA

should

not

rule

out

an

enzyme

function

as

absolutely

as

first

appeared

An

alternative

possibility

to

the

biosynthetic

pathway

we

have

outlined

-LRB-

Fig.

-RRB-

is

that

WbmF

does

in

fact

catalyse

either

oxidation

or

reduction

as

well

as

the

in

which

case

the

third

SDR

may

participate

in

O-antigen

expression

by

performing

a

function

in

the

synthesis

of

the

complicated

sugar

residue

that

adorns

the

nonreducing

terminus

of

the

The

nonredox

role

we

propose

for

WbmF

is

consistent

however

with

the

lack

of

the

conserved

SDR

triad

in

this

enzyme

and

if

this

protein

operates

purely

as

a

this

may

also

suggest

a

mechanism

by

which

the

wbmF

mutant

is

able

to

express

O

antigen

albeit

in

greatly

reduced

amounts

The

product

of

the

initial

oxidation

reaction

is

activated

for

proton

exchange

α

to

the

keto

group

and

it

may

be

that

racemisation

at

these

positions

occurs

at

a

biologically

significant

rate

without

an

absolute

requirement

for

enzyme

catalysis

Recent

structural

studies

of

the

in

the

dTDP-rhamnose

pathway

RmlC

suggest

that

the

rate-limiting

step

in

its

reaction

involves

a

transition

state

in

which

the

anomeric

linkage

is

in

the

disfavoured

equatorial

position

-LRB-

which

is

also

adopted

by

the

reaction

product

-RRB-

and

that

in

order

to

avoid

steric

clashes

and

satisfy

the

requirement

for

axial

proton

abstraction

the

reaction

mechanism

requires

a

twist-boat

conformation

such

as

that

proposed

in

the

GME

reaction

Binding

of

substrate

in

a

equatorial

conformation

may

be

part

of

the

way

that

RmlC

reduces

kinetic

barriers

to

this

reaction

For

the

same

reasons

the

product

of

the

proposed

WbmF-catalysed

will

have

the

nucleotide

attached

to

the

l-GalNAcNAcA

in

the

equatorial

position

in

order

to

avoid

clashes

This

transformation

therefore

involves

considerable

kinetic

and

thermodynamic

barriers

and

will

be

slow

without

enzyme

catalysis

However

if

sufficient

starting

material

is

generated

by

the

upstream

oxidase

and

the

product

is

efficiently

and

selectively

removed

by

the

downstream

reductase

the

flux

through

this

bottleneck

may

be

sufficient

to

allow

O-chain

synthesis

Furthermore

the

slower

rate

resulting

from

mutation

of

the

wbmF

gene

may

make

relatively

little

impact

on

O-antigen

expression

levels

if

this

conversion

does

not

usually

comprise

the

rate-limiting

step

for

O-antigen

synthesis

A

similar

effect

may

operate

in

the

dTDP-l-noviose

pathway

in

Streptomyces

spheroides

where

observation

of

a

naturally

occurring

l-rhamnoside

analogue

of

shows

that

of

the

dTDP-noviose

precursor

-LRB-

-RRB-

does

occur

despite

the

fact

that

in

vitro

the

catalysed

by

NovW

has

a

lower

kcat

than

-LRB-

discussed

in

Ref

-RRB-

In

conclusion

X-ray

crystallography

of

WbmF

WbmG

and

WbmH

enabled

definition

of

these

enzymes

NAD-cofactor

preference

and

the

substrate

nucleotide

specificity

We

have

been

able

to

distinguish

the

likely

function

of

WbmF

from

WbmG

and

WbmH

but

can

not

differentiate

the

roles

of

WbmG

and

WbmH

In

order

to

achieve

this

these

proteins

must

be

characterised

in

vitro

This

will

require

chemical

synthesis

of

and

is

beyond

the

scope

of

the

present

study

Subject

to

future

experimental

verification

of

predictions

we

have

made

our

data

illustrate

the

usefulness

of

structural

studies

for

investigating

such

challenging

problems

While

they

also

show

why

this

methodology

can

not

universally

supplant

direct

in

vitro

characterisation

this

kind

of

structural

analysis

will

prove

essential

for

researchers

to

make

full

use

of

the

gene

databases

Materials

and

Methods

Bacterial

strains

plasmids

and

culture

conditions

Bacterial

strains

used

in

this

study

are

described

in

Table

Bordetella

was

grown

on

Bordet

Gengou

agar

-LRB-

Difco

-RRB-

supplemented

with

defibrinated

horse

blood

-LRB-

TCS

Cellworks

Ltd

-RRB-

E.

coli

was

cultured

in

Luria

Bertani

-LRB-

LB

-RRB-

broth

or

on

LB

agar

All

strains

were

incubated

at

°C

and

ampicillin

-LRB-

μg

ml

-RRB-

kanamycin

-LRB-

μg

ml

-RRB-

tetracycline

-LRB-

μg

ml

for

E.

coli

μg

ml

for

B.

bronchiseptica

-RRB-

or

streptomycin

-LRB-

μg

ml

-RRB-

were

added

where

required

Suicide

plasmids

were

based

on

the

host-restricted

and

broad

host

range

shuttle

vectors

were

based

on

a

Kmr

derivative

of

For

preparation

of

LPS

B.

bronchiseptica

was

grown

in

medium

supplemented

with

mM

as

this

maximises

O-antigen

expression

in

this

strain

DNA

methods

Standard

methods

were

used

for

DNA

manipulations

Oligonucleotides

were

supplied

by

Sigma-Genosys

PCR

was

performed

with

template

from

boiled

and

Taq

DNA

polymerase

-LRB-

Promega

-RRB-

or

KOD

Hot

Start

DNA

polymerase

-LRB-

Novagen

-RRB-

Construction

of

wbmF

wbmG

and

wbmH

mutants

The

multiple

cloning

site

-LRB-

MCS

-RRB-

from

pBluescript

II

SK

-LRB-

Stratagene

-RRB-

was

excised

using

SacI

and

KpnI

blunt-ended

and

ligated

into

SmaI-cut

to

generate

the

MCS-containing

suicide

vector

pEXMCS

The

wbmF

allelic

exchange

construct

was

prepared

as

follows

The

wbmF

gene

in

the

plasmid

-LRB-

a

clone

generated

in

the

B.

bronchiseptica

genome

sequencing

project

-RRB-

was

disrupted

by

insertion

of

a

blunt-ended

tetracycline-resistance

-LRB-

Tc

-RRB-

cassette

into

the

internal

NsiI

site

The

mutant

allele

was

excised

using

and

XbaI

blunt

ended

and

then

cloned

into

EcoRV-digested

pEXMCS

For

wbmG

the

gene

was

amplified

using

primers

-LRB-

XbaI

site

underlined

-RRB-

and

and

the

PCR

product

was

topoisomerase

cloned

into

using

a

TOPO-cloning

kit

-LRB-

Invitrogen

-RRB-

wbmG

was

excised

using

XbaI

and

cloned

into

the

XbaI

site

in

pEXMCS

The

wbmG

gene

was

then

disrupted

by

cloning

a

blunt-ended

Tc

cassette

into

the

internal

site

The

wbmH

mutant

allele

was

obtained

by

in

vitro

transposon-mediated

mutagenesis

of

the

wbm

locus-containing

cosmid

-LRB-

accession

number

-RRB-

using

an

insertion

kit

-LRB-

Epicentre

-RRB-

The

transposon

plus

flanking

wbmH

DNA

was

cut

out

by

partial

digestion

with

AluI

and

ligated

into

SmaI-cut

Allelic

exchange

constructs

were

electroporated

into

E.

coli

and

transferred

to

B.

bronchiseptica

by

conjugation

with

E.

coli

as

donor

Loss

of

the

plasmid-encoded

sacB

gene

in

allelic

exchange

mutagenesis

of

B.

bronchiseptica

was

selected

for

by

growth

on

LB

agar

with

reduced

salt

concentration

and

supplemented

with

-LRB-

w/v

-RRB-

sucrose

Complementation

of

mutations

The

B.

bronchiseptica

flaA

promoter

was

amplified

using

primers

-LRB-

XbaI

site

underlined

-RRB-

and

-LRB-

BamHI

and

NdeI

sites

underlined

-RRB-

and

cloned

into

the

XbaI

and

BamHI

sites

in

to

generate

the

vector

pCompEmpty

wbmF

was

amplified

using

primers

-LRB-

NdeI

site

underlined

-RRB-

and

-LRB-

HindIII

site

underlined

-RRB-

and

cloned

into

pCompEmpty

using

NdeI

and

HindIII

to

generate

the

wbmF

complementation

vector

pCompF

wbmG

was

PCR

amplified

using

primers

and

and

cloned

into

pCompEmpty

using

NdeI

and

HindIII

generating

pCompG

wbmH

was

amplified

using

and

and

cloned

into

pCompEmpty

using

NdeI

and

HindIII

generating

pCompH

Shuttle

vectors

were

moved

into

B.

bronchiseptica

by

conjugation

with

E.

coli

as

and

trans-acting

transfer

functions

provided

by

E.

coli

as

helper

SDS-PAGE

silver

stain

and

immunoblot

analysis

of

LPS

LPS

was

obtained

from

B.

bronchiseptica

using

a

modification

of

the

method

of

Hitchcock

and

as

has

been

described

SDS-PAGE

of

LPS

was

performed

using

Novex

precast

tricine

gels

-LRB-

Invitrogen

-RRB-

LPS

was

oxidised

in-gel

with

periodic

and

visualised

with

the

Silver

Stain

Plus

kit

-LRB-

BioRad

-RRB-

Western

blotting

was

performed

as

previously

using

the

monoclonal

antibody

that

specifically

binds

the

O-antigen-containing

LPS

of

B.

bronchiseptica

strain

X-ray

crystallography

Protein

production

purification

crystallisation

and

data

collection

were

performed

as

described

Data

were

processed

using

DENZO

and

SCALEPACK

-LRB-

version

-RRB-

Data

were

truncated

and

converted

to

structure

factors

using

from

the

Suite

Molecular

replacement

was

carried

out

using

as

described

For

the

initial

WbmG

structure

phases

were

determined

experimentally

from

selenomethionine-labelled

protein

peak

inflection

point

and

high-energy

remote

data

were

used

to

determine

initial

phases

using

the

PHENIX

package

Model

building

was

carried

out

using

COOT

and

refinement

was

performed

with

REFMAC

version

Initial

models

were

improved

using

ARP/wARP

version

where

the

data

quality

permitted

The

was

used

where

appropriate

TLS

parameters

were

assigned

-LRB-

where

used

-RRB-

using

the

TLSMD

server

was

used

to

perform

simulated

annealing

Structures

were

validated

using

COOT

PROCHECK

WHATCHECK

and

RAMPAGE

Table

provides

a

summary

of

the

crystallographic

statistics

Protein

structures

were

analysed

using

Pymol

COOT

and

Surface

area

calculations

were

performed

using

the

Richards

Rolling

Probe

method

More

extensive

details

of

the

crystallographic

methods

are

provided

in

the

Supplementary

Data

Modelling

of

putative

substrate

into

WbmF

structure

Coordinates

and

parameters

for

the

derivative

of

UDP-2,3-dideoxy-2,3-diacetamido-d-mannuronic

acid

were

prepared

using

the

monomer

library

sketcher

with

refinement

in

REFMAC

An

initial

structure

was

prepared

by

modelling

the

proposed

substrate

into

the

structure

of

WbmF

co-crystallised

with

UDP

in

COOT

The

observed

density

was

used

to

place

the

atoms

present

in

UMP

and

to

place

sugar

atoms

in

positions

occupied

by

water

or

glycerol

in

the

observed

structure

Different

orientations

were

used

in

the

two

molecules

in

the

asymmetric

unit

to

increase

the

sampling

of

conformational

space

The

structure

of

WbmF

co-crystallised

with

UDP

with

placed

in

was

truncated

to

include

only

protein

NAD

and

atoms

This

was

used

as

an

input

to

MODELLER

version

to

determine

an

optimised

structure

for

the

complex

with

the

sugar

nucleotide

After

modelling

the

ligand

coordinates

were

regularised

in

COOT

to

correct

minor

distortions

Ten

modelling

trials

were

performed

with

different

seeds

and

the

best

was

selected

by

eye

on

the

basis

of

geometry

agreement

with

the

determined

structures

and

lack

of

bad

interactions

Protein

Data

Bank

accession

codes

X-ray

amplitudes

phases

and

the

derived

atomic

coordinates

have

been

deposited

with

the

PDB

under

the

accession

codes

and

O O O O O O O O O O O O O B O O O O O B O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

blast, basic local alignment search tool