Lennarz W.J., Lane M.D. (eds.) Encyclopedia of Biological Chemistry. Four-Volume Set . V. 4

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FURTHER READING

Arata, Y., Baleja, J. D., and Forgac, M. (2002). Localization of subunits

D, E and G in the yeast V-ATPase complex using cysteine-mediated

cross-linking to subunit B. Biochemistry 41, 11301– 11307.

Bowman, B. J., and Bowman, E. J. (2002). Mutations in subunit c

of the V-ATPase confer resistance to baﬁlomycin and identify

a conserved antibiotic binding site. J. Biol. Chem. 277,

3965–3972.

Nishi, T., and Forgac, M. (2002). The vacuolar (H

)-ATPases:

Nature’s most versatile proton pumps. Nat. Rev. Mol. Cell Biol.

3, 94–103.

Smardon, A. M., Tarsio, M., and Kane, P. M. (2002). The RAVE

complex is essential for stable assembly of the yeast V-ATPase.

J. Biol. Chem. 277, 13831–13839.

Toyomura, T., Oka, T., Yamaguchi, C., Wada, Y., and Futai, M.

(2000). Three subunit a isoforms of mouse vacuolar (H

)-ATPase.

Preferential expression of the a3 isoform during osteoclast

differentiation. J. Biol. Chem. 275, 8760– 8765.

BIOGRAPHY

Michael Forgac is a Professor in the Department of Physiology at Tufts

University School of Medicine. His principal research interest is in the

structure, mechanism, and regulation of the V-ATPases. He received his

B.S. in biology and chemistry from Caltech in 1976 and his Ph.D. in

Biochemistry and Molecular Biology from Harvard in 1981. He is

author of over eighty publications and a recipient of a MERIT Award

from the National Institutes of Health.

V-ATPases 353

Vitamin A (Retinoids)

Joseph L. Napoli

University of California, Berkeley, CA, USA

Vertebrates require vitamin A, all-trans-retinol (atROH), for

vision, fertility, embryogenesis, growth, and optimum neuro

and immune function. atROH generates the visual pigment,

11-cis-retinal, and the humoral effector all-trans-retinoic

acid (atRA). atROH transport and metabolism relies on

serum and cellular chaperones (binding proteins) to maxi-

mize efﬁciency and impose speciﬁcity. Cleavage of caroten-

oids in the intestinal mucosa by carotenoid monooxygenase

produces all-trans-retinal. CRBP(II) binds all-trans-retinal

and dietary retinol, and allows only reduction and

esteriﬁcationintoall-trans-retinyl esters (atRE). atRE

storage occurs mostly in liver stellate cells. Liver only

releases atROH bound with serum retinol binding protein,

sRBP. In extra-intestinal tissues, cellular retinol binding-

protein (CRBP) binds atROH and allows re-esteriﬁcation. In

the retinal pigment epithelium, the binding-protein RPE65

facilitates concerted atRE hydrolysis and conversion into

11-cis-retinol, catalyzed by an isomerohydrolase. Short-

chain dehydrogenase/reductases (SDR) convert 11-cis-retinol

into 11-cis-retinal. 11-Cis-retinal crosses the interphoto-

receptor matrix and enters the rod outer segments, where it

binds covalently with opsin to form rhodopsin. Light

isomerizes 11-cis-retinal into all-trans-retinal, changing the

conformation of rhodopsin and generating a nerve impulse.

The transporter ABCR aids leaching of all-trans-retinal

from rhodopsin. Other SDR reduce all-trans-retinal into

atROH. atROH crosses the interphotoreceptor space,

rebinds with CRBP in the retinal pigment epithelium, and

undergoes re-esteriﬁcation. atRA biogeneration takes a

different path. atROH, from hydrolysis of atRE by retinyl

ester hydrolase or from serum, binds with CRBP and

undergoes dehydrogenation by microsomal SDR. The all-

trans-retinal produced undergoes dehydrogenation into

atRA, catalyzed by retinal dehydrogenases. CRABP(II)

delivers atRA to retinoic acid receptors, whereas the

complex CRABP-atRA has higher enzymatic efﬁciency for

atRA degradation than does free atRA. Cytochromes P-450

initiate atRA degradation through C4 and C18 hydroxy-

lation and 5,6-epoxydation. Primary regulators of atROH

homeostasis include apo-CRBP, which inhibits esteriﬁcation

of atROH and accelerates hydrolysis of atRE, and atRA,

which induces its own catabolism.

Retinoids and their Functions

The term retinoid refers to compounds, both naturally

occurring and synthetic, with vitamin A activity: vitamin

A denotes the organic compound all-trans-retinol

(atROH). All vertebrates require vitamin A/atROH for

vision, fertility, embryogenesis, and growth. atROH does

not support the physiological functions attributed to

vitamin A; rather it acts as precursor for biosynthesis of

retinoids directly responsible for producing “vitamin A

activity”. These atROH metabolites include 11-cis-

retinal, the cofactor covalently bound with opsin to

form the visual pigment rhodopsin, and all-trans-retinoic

acid (atRA), the humoral effector of the non-visual,

systemic functions attributed to vitamin A. These sys-

temic functions include: controlling the differentiation

programs of all epithelia, and of stem cells in the skin,

nervous system, bone, immune system, hemopoietic

system; acting as a tumor suppressor; and regulating

apoptosis. Speciﬁc non-visual effects of retinoids include

memory formation, immune responsivity, stress adap-

tation, and cell fate determination. atRA activates three

nuclear receptors, RAR

and

, and thereby controls

the expression of hundreds of genes. atRA, or perhaps

other retinoids, may also function through non-genomic

mechanisms, but the research is in its infancy.

The Chemistry of atROH

Generation, Storage, and

Metabolic Activation

Carotenoids with at least one

-ionone ring, especially

-carotene, provide the major retinoid precursors in

most diets. Oxidative central cleavage by a plasma-

membrane associated, but soluble, carotene 15,15

monooxygenase (CMO) produces all-trans-retinal—a

transient intermediate that occurs in very low concen-

trations outside of the neural retinal. Microsomal

retinal reductases, RRD, members of the short-chain

dehydrogenase/reductase (SDR) gene family, catalyze

reduction of all-trans-retinal into atROH, which then

undergoes esteriﬁcation into all-trans-retinyl esters

(atRE), predominantly palmitate, by microsomal

lecithin:retinol acyltransferase (LRAT). Chylomicrons

carry atRE into circulation and chylomicron remnants

deliver them to liver, where ultimately, most are stored

in stellate cells.

In the visual cycle, atRE undergo concerted

hydrolysis– isomerization into 11-cis-retinol by a micro-

somal isomerohydrolase (IMH), followed by dehydro-

genation into 11-cis-retinal (SDR). 11-Cis-retinal forms

a Schiff’s base with a lysine residue in the protein opsin

to form rhodopsin. When light strikes the neural retinal,

11-cis-retinal in rhodopsin undergoes cis to trans-

isomerization, causing a conformation change that

initiates nerve impulses and releases the newly formed

all-trans-retinal. Reduction of all-trans-retinal into

atROH (microsomal SDR) and re-esteriﬁcation

(LRAT) into atRE completes the visual cycle.

Activation of atROH into atRA uses the same

intermediate used in the visual cycle, all-trans-retinal,

but relies on a metabolically distinct route. atROH,

either from blood or from hydrolysis of retinyl esters

by microsomal retinyl ester hydrolase (REH), undergoes

reversible dehydrogenation into all-trans-retinal, cata-

lyzed primarily by microsomal retinol dehydrogenases,

RDH, also members of the SDR gene family. In contrast

to the comparatively high concentrations of retinals that

allow the visual cycle to function, all-trans-retinal

concentrations during atRA biosynthesis are kept low

by reduction (back reaction of RDH and reduction

by microsomal and peroxisomal reductases), and by

irreversible dehydrogenation into atRA, catalyzed by

soluble, , 54 kDa, high V

max

retinal dehydrogenases

(RALDH), members of the aldehyde dehydrogenase

(ALDH) gene family. atRA isomers occur in vivo, such

as 13-cis-RA and 9,13-di-cis-RA, but their signiﬁcance

and source(s) remain unclear.

These straightforward reactions offer complex oppor-

tunities for physiological regulation, owing to compart-

mentalization, distinct enzymes catalyzing each

direction of chemically reversible reactions (e.g. dehy-

drogenation/reduction of retinol/retinal; esteriﬁcation/

hydrolysis of atROH/atRE), cell-distinct expression

patterns, and multiple homologs/paralogs that catalyze

several reactions. For example, at least four reductases

have been identiﬁed, which belong to the SDR gene

family, Rrd (peroxisomal) and RalR1 outside of the eye,

and retSDR and prRDH, in the neural retina. At least

three RDH, also SDR, have been identiﬁed in the rat:

Rodh1, Rodh2 and Rodh3. Four Raldh have been

identiﬁed in human, rat and mouse: Raldh1, 2, 3, 4 (aka

ALDH 1A1, 1A2, 1A6 and 8A1). These constitute a

complex enzyme system for absorbing and storing

vitamin A, maintaining atRA homeostasis, and recycling

vitamin A in the visual cycle.

Retinoid Binding-Proteins

and their Contributions to

Retinoid Homeostasis

Processing of dietary retinoids and retinoid precursors

and biogeneration of active retinoids relies on serum

and cellular chaperones for efﬁcient and speciﬁc

retinoid use, as demonstrated by studies in vitro and

the consequences of gene knockouts and/or naturally

occurring mutations.

Liver and other tissues synthesize a retinol binding-

protein, sRBP, a member of the lipocalin gene family.

atROH egress from liver requires sRBP, and sRBP-

atROH represents the major form of vitamin A in serum.

sRBP circulates as a complex with a tetramer of

transthyretin, which protects the , 20 kDa sRBP from

kidney ﬁltration. The mechanism of atROH delivery

from sRBP into cells has not been established. Some data

suggest a membrane receptor, other data indicate that

cellular retinol binding-protein(s) draw atROH from

sRBP through the membrane. A third hypothesis would

have a sRBP receptor mainly in eye, the major site of

vitamin A consumption. The sRBP null mouse seems

phenotypically normal, except for impaired vision after

weaning. Feeding a vitamin A-adequate diet for months

restores vision. Although the eye relies on sRBP for

efﬁcient atROH uptake, atROH obtained from post-

prandial lipoprotein delivery can substitute, at least

under laboratory conditions. Interestingly, atRA serum

levels increase in the sRBP null mouse, indicating that

serum delivery of atRA to tissues helps compensate for

impaired atROH delivery.

Binding-proteins channel retinoid intermediates

through the series of reactions that constitute the visual

cycle (Figure 1). Mice null in the retinal pigment protein

(RPE) RPE65, for example, cannot produce 11-cis-

retinoids, consistent with an RPE65 function of binding

the hydrophobic atRE (K

value , 20 pM), and accel-

erating their mobilization and delivery to the next step,

acyl hydrolysis and C11 isomerization by the IMH.

RPE65 belongs to the same gene family as the

carotenoid-metabolizing enzyme CMO (the mouse

proteins have only 37% amino acid identity, however),

suggesting a gene family devoted to transport/meta-

bolism of hydrophobic substances. The IMH product,

11-cis-retinol, undergoes sequestration in the RPE by the

36 kDa cytosolic cellular retinal binding-protein

(CRALBP), a member of the gene family that includes

the

-tocopherol transfer protein, TTP. CRALBP facili-

tates dehydrogenation of 11-cis-retinol into 11-cis-

retinal, and drives forward the trans to cis isomerization.

VITAMIN A (RETINOIDS) 355

Mutations in human CRALBP cause night blindness and

photoreceptor degeneration. RDH4 and RDH5, respect-

ively, represent the murine and human 11-cis-retinol

dehydrogenase, which interacts with and/or accepts

substrate from CRALBP. Mutations in RDH5 are

associated with the rare, autosomal recessive disease

fundus albipunctatus, i.e. night blindness from delayed

photopigment regeneration. Lack of total blindness in

the case of the RDH5 mutation, indicates that SDR in

addition to RDH4/5 contribute to 11-cis-retinal biosyn-

thesis. 11-Cis-retinal tranverses the interphotoreceptor

matrix by an unknown mechanism and covalently binds

with opsin to form rhodopsin in the rod outer segment

(ROS). After the action of light, an ATP-dependent

transporter, ABCR, facilitates release of all-trans-retinal

from rhodopsin. prRDH then reduces the all-trans-

retinal into atROH, which then travels back through the

interphotoreceptor space to the RPE.

Several additional cellular retinoid binding-proteins

occur. The four understood to be the most important,

other than those mentioned above with respect to the

visual cycle, include CRBP, CRBP(II), CRABP, and

CRABP(II) (Table I). All vertebrates express all four,

with well-conserved amino acid sequences among

orthologs. All four are high-afﬁnity, soluble, and speciﬁc

for their ligands. CRBP binds atROH, and closely

related compounds such as 3,4-didehydro-atROH, and

all-trans-retinal to a lesser extent, and discriminate

against cis-retinols and atRA. CRABP binds atRA,

metabolites such as 4-OH-atRA, and discriminates

against cis-RAs and atROH. These , 15 kDa globular

proteins belong to the intracellular lipid binding-protein

(iLBP) gene family, which includes the various fatty acid

binding-proteins.

The cellular retinol/retinal binding-proteins enclose

atROH and all-trans-retinal with the hydroxyl/aldehyde

function, crucial to metabolic activation, sheltered

inside. These two proteins apparently confer selective

advantage to vertebrates by enhancing efﬁciency of

sequestering, transporting and storing vitamin A, and

limiting its catabolism. Vitamin A absorption and

biosynthesis in the intestine relies on CRBP(II). CRBP(II)

systemic, humoral

vitamin A actions

atRA

CRBP-atROH

LRAT

(–)

(CH

)

all-

trans

-retinyl ester

atROH

CMO

b-carotene

15′

RRD RDH1

all-

trans

-retinal

RALDH

RXR-RAR

CRABP(II)

CRABP-atRA

catabolites

CYP

(+)

CRABP(II)-atRA

RXR-RAR-atRA

CRABP

IMH

CRALBP

RPE65

RPE65-atRE

CRALBP-11-

cis

-ROH

RDH5

11-

cis

-retinal

CRALBP

vision

opsin

rhodopsin

atRCHO

ABCR

prRDH

apo-CRBP

(+)

REH

ROS

RPE

FIGURE 1 Major paths of retinoid metabolism. The white background designates reactions of retinol homeostasis and activation into atRA in

the liver and extra-hepatic vitamin A target-tissues. The light gray background depicts reactions that occur both in extra-ocular tissues and in the

retinal pigment epithelium (RPE) of the neural retina. The light yellow background designates reactions that occur in the RPE and in the rod outer

segments (ROS). Numbers indicate the positions of trans to cis isomerization (C11), the positions of hydroxylations that occur during catabolism of

atRA (C4 and C18), and the positions of atRA isomers of physiological interest (C9, C13). The dotted lines indicate the direct actions of apo-CRBP

in regulating atRE hydrolysis and atROH esteriﬁcation. The dashed-dotted line indicates transcriptional induction of cytochromes P-450 (CYP) by

atRA. For simplicity, CRBP(II) is not shown.

356 VITAMIN A (RETINOIDS)

null mice pups die within 24 hours after birth, when

delivered by dams fed a diet marginal in vitamin A

content. CRBP(II) contributes , 1% of the soluble

protein to the intestinal enterocyte—an indication of a

mass-action function to sequester newly synthesized

(from carotenoids) all-trans-retinal or newly absorbed

dietary atROH. In vitro, all-trans-retinal bound with

CRBP(II) undergoes reduction readily, but neither it nor

bound atROH undergoes dehydrogenation. This would

limit production of atRA, and other metabolism, from

the bolus of all-trans-retinal produced during carotenoid

metabolism. In the intestine, the esterifying enzyme

LRAT recognizes atROH bound with CRBP(II) to

produce atRE for incorporation into chylomicrons.

Thus, CRBP(II) likely aids atROH uptake and chaper-

ones the products of carotenoid metabolism down the

pathway to atRE to enhance efﬁciency of retinoid

recovery from the diet.

Extra-intestinal vitamin A uptake and storage relies

on CRBP. CRBP-null mice seem morphologically nor-

mal, but eliminate atRE 6-fold faster than wild-type

mice, and may sequester/esterify atROH less efﬁciently.

Clearly, efﬁcient use of atROH in vivo depends on the

chaperone. Strikingly, CRBP has a K

value for atROH

far lower than the atROH concentration in tissues

(1–30 mM), and CRBP concentrations exceed atROH

concentrations, where measured. The law of mass action

predicts from these data that non-esteriﬁed atROH

would occur nearly exclusively in the CRBP-bound

state, assuming no alternative high-afﬁnity or very high-

capacity acceptors. Alternatives to CRBP exert limited

inﬂuence in vivo, because isolation of CRBP from

animal tissues by traditional (time-consuming and

containing membranes, lipids and lipid vesicles) bio-

chemical techniques (tissue homogenization, centrifu-

gation and several types of column chromatography)

produces largely holo-protein. Evidently, the capacity of

membranes and other potential acceptors to sequester

atROH does not overwhelm the ability of CRBP to

sequester atROH. Like CRBP(II), LRAT can access

atROH bound with CRBP to produce atRE. Ultimately,

liver stellate cells accumulate most of the atRE.

CRBP seems necessary for retinoid transfer from

hepatocytes to stellate cells, because the CRBP null

mouse does not accumulate atRE in stellate cells.

atROH sequestering within CRBP, the need to store

retinol as esters, and the need for atRA biosynthesis

during speciﬁc times at speciﬁc loci, suggest that transfer

of atROH for metabolism might depend on relation-

ships between metabolizing enzymes and CRBP. Like

CRBP(II), CRBP allows esteriﬁcation of bound atROH,

but in contrast to CRBP(II), CRBP also allows dehy-

drogenation of atROH. The CRBP–atROH complex

shows Michaelis–Menton relationships with atRE

formation by LRAT and atROH dehydrogenation by

RDH. This relationship with the microsomal RDH is

maintained even with changes in the CRBP/atROH ratio

(provided the CRBP concentration exceeds the atROH

concentration). This eliminates a “free diffusion”

mechanism of transfer from the complex to select

enzymes. Speciﬁc crosslinking of holo-CRBP with both

RDH and LRAT conﬁrms close proximity of CRBP and

these two enzymes. Additionally, a single mutation in an

exterior residue of CRBP (L35A) reduces the Vm of

atROH dehydrogenation by microsomes, but does not

alter the K

, or the K

of atROH binding to CRBP,

consistent with conservation of exterior residues that aid

transfer of atROH from CRBP to enzymes. Obviously,

atRE and atRA biosynthesis in vivo occurs in the

absence of CRBP, as indicated by the lack of morpho-

logical pathology in the CRBP null mouse, and their

ability to sequester esters. This was predicted by the

experiments in vitro, which showed that neither RDH

nor LRAT require presentation of atROH by CRBP. Not

surprisingly, the enzymes’ active sites recognize their

substrates in the absence of CRBP. CRBP operates as a

chaperone, which restricts atROH metabolism to select

enzymes, and seems required only for efﬁcient atROH

use in vivo. The ability of LRAT and RDH to access

retinol from CRBP addresses the issue of how atROH

would undergo efﬁcient metabolism in the face of

limited diffusion from the binding protein.

RALDH catalyze the irreversible conversion of

atRCHO into atRA in the presence of CRBP, and also

TABLE I

Major Extra-ocular Retinoid Binding-Proteins

Retinoid binding-protein Ligand(s) K

(nM) Post-embryonic distribution

CRBP (cellular retinol binding-protein, type I) atROH , 0.1 Nearly ubiquitous (low in intestine)

All-trans-retinal 10–50

CRBP(II) (cellular retinol binding-protein, type II) atROH 10–50 Intestine, neonatal liver

All-trans-retinal 10–50

CRABP (cellular retinoic acid binding-protein, type I) atRA 0.4 Widespread

CRABP(II) (cellular retinoic acid binding-protein, type II) atRA 2 Limited (inducible?) (skin, uterus, ovary)

VITAMIN A (RETINOIDS) 357

can use atRCHO generated in situ from CRBP-atROH

and RDH, or in cells presented with atROH and

transfected with RDH and RALDH. In the rat,

RALDH1 and RALDH2 have differing but overlaping

expression patterns, and respond differently to changes

in atROH status. This suggests a purpose for more than

one—precise control over atROH use and atRA

generation. The RALDH1 null mouse remains fertile

and healthy, but may have decreased ability to produce

atRA in the liver. The RALDH2 null mouse dies in utero

at midgestation, demonstrating its unique contribution

to atRA biosynthesis during embryogenesis. The situ-

ation may differ in the adult, as testes express RALDH2

strongly, but RALDH1 prevails outside of the testis. The

RALDH3 null mouse dies during suckling from an

obstruction in the nose. Apparently, RALDH can

compensate for each other after critical developmental

milestones.

A CRBP(III) has been detected in mouse heart and

skeletal muscle, which express little or no CRBP or

CRBP(II), but has not been detected in other retinoid

target tissues, such as liver, kidney, brain, etc. CRBP(III)

seems to bind about equally well with atROH, 9-cis-

retinol and 13-cis-retinol, but with much lower afﬁnity

that either CRBP or CRBP(II) (K

, 80–110 nM).

Humans express yet another CRBP, originally referred

to as CRBP(III), but distinct from mouse CRBP(III), and

therefore CRBP(IV). CRBP(IV) mRNA is much more

abundant in human liver and intestine than CRBP

mRNA, but the mouse does not encode a complete

CRBP(IV) gene. CRBP(IV) binds atROH with a K

value

of , 60 nM, and does not bind cis-isomers. The precise

functions of CRBP(III) and CRBP(IV) have not been

clariﬁed, and unlike CRBP and CRABP, their endogen-

ous ligands have not been established.

The atRA binding-proteins, CRABP and CRABP(II),

do not have well-deﬁned functions. Doubly null mice

have a , 4-fold higher rate of death from unknown

causes by 6 weeks after birth than wild-type, but the

survivors appear normal, with one exception. The

doubly null mouse, as well as the CRABP(II)-only null

mouse, show 83% and 45%, respectively, incidence of

a small outgrowth anomaly on the post-axial side of

digit ﬁve, predominantly in the forelimbs. Mice doubly

null in CRABP and CRABP(II) do not exhibit enhanced

sensitivity to exogenous atRA, suggesting that the

binding-proteins do not protect against atRA toxicity.

In contrast to CRBP, both CRABP and CRABP(II)

allow projection of the

-ionone ring of their ligand.

Signiﬁcantly, the ﬁrst reactions of atRA degradation

occur at these comparatively accessible sites, i.e. C4

and C18. Presenting atRA to microsomes bound

with CRABP enhances kinetic efﬁciency (K

cat

)of

catabolism 7-fold. There seems to be little doubt

that CRABP sequesters atRA: delivering the seques-

tered atRA for efﬁcient catabolism seems a logical

mechanism to discharge the ligand without releasing it

back into the cell. Unfortunately, this insight doesn’t

reveal the primary purpose for CRABP impounding

atRA in the ﬁrst place, although CRABP tends to

express in cells different from those that express CRBP

and CRABP(II). CRABP(II), but not CRABP, seems to

deliver atRA to RAR, via a transfer that does not

require free diffusion. This would complete the

chaperoning of atROH on its journey from atRE

through atRA biogenesis to nuclear localization.

Many other enzymes, including medium-change

alcohol dehydrogenases, and aldo-keto reductases,

metabolize retinoids in vitro, which seems to conﬁrm

the need for evolution of CRBP to protect the sparse and

valuable vitamin A from clearance as a “xenobiotic”.

These enzymes do not access atROH bound with CRBP.

Neither the ADHI-null deermouse, a natural mutant,

nor mice made null in ADHI, ADHIV, or ADHIII or

doubly null in ADHI and ADHIV, present with vitamin

A deﬁciency symptoms, revealing no inability to activate

atROH. ADHI-null mice do show decreased ability to

convert a very large dose (50 mg kg

) of atROH into

atRA. Such a dose has no natural equivalent: the results

indicate only that determination can overwhelm physio-

logical chaperones.

Other Naturally

Occurring Retinoids

Discrete loci, such as skin and the chick limb bud,

synthesize 3,4-didehydro-atRA. 3,4-Didehydro-atRA

binds retinoic acid receptors with afﬁnity similar to that

of atRA. The purpose has not been clariﬁed of creating a

signaling molecule that functions as atRA in specialized

loci that also biosynthesize atRA. Although 9-cis-RA

was reported as an activated retinoid, it is virtually

undetectable in vivo: its putative function as a physio-

logical ligand that controls RXR remains uncertain.

Control of Vitamin A Homeostasis

The primary regulators of retinoid homeostasis appear

to be apo-CRBP and atRA. apo-CRBP inhibits LRAT

and stimulates retinyl ester hydrolysis. Thus, the

direction of ﬂux into or out of atRE would reﬂect

the ratio apo-CRBP/holo-CRBP, which would reﬂect the

atROH status of the cell. atRA may serve as a signal to

liver to release atROH into the serum. Little has been

revealed about humoral regulation of atRA biosynthesis,

but estrogen and PGE, increase and decrease, respect-

ively, atRA biosynthesis in cultured cells. The meta-

bolism of atRA limits its activity; conversely, inhibitors

of atRA metabolism enhance atRA potency. atRA

358

VITAMIN A (RETINOIDS)

induces its own oxidative metabolism into 5,6-epoxy-

atRA, 18-hydroxy-atRA, and 4-hydroxy-atRA, through

inducing cytochrome P-450 s (CYP). CYP26A1 and

CYP2C39 appear to have major functions in the

catalysis of atRA catabolism. CYP26A1 null mice die

in mid to late gestation with serious morphological

defects. Two other CYP, 26B1 and 26C1, also catabolize

atRA, but their precise function has not been clariﬁed.

Many other CYP reportedly catabolize atRA (e.g.

CYP1A1/2, CYP2A6, CYP2C8/9, CYP2E1,

CYPP3A4/5), but atRA does not induce these isoforms

and most have inefﬁcient kinetics with atRA in vitro.

PPAR

induces transcription of CMO, CRBP,

CRBP(II), LRAT, and RAR

, suggesting correlation

between vitamin A homeostasis and general lipid

metabolism, whereas LRAT and CRBP are among the

numerous genes induced by atRA. This action of atRA

may represent a “housekeeping” function, rather than

acute control over retinoid homeostasis.

Several xenobiotics, including ethanol and poly-

chlorinated aromatic hydrocarbons reduce atRE stores,

possibly through enhancing atRA catabolism by indu-

cing CYP: the polychlorinated hydrocarbons, such as

dioxin, function through the AH receptor to decrease

atRE stores.

Clinical Uses of Retinoids

Numerous studies have correlated vitamin A insufﬁ-

ciency in laboratory animals with increased incidence

of spontaneous and carcinogen-induced cancer. Chemo-

preventive trials in humans show some promise for

retinoids in actinic keratoses, oral premalignant lesions,

laryngeal leukoplakia, and cervical dysplasia. The FDA

has approved retinoids for acute promyelocytic

leukemia and in non-life-threatening diseases such

as cystic acne and psoriasis. Retinoids also provide

the active ingredients in agents to treat sun-/age-

damaged skin.

The WHO recognizes vitamin A-deﬁciency as a

mortality factor for childhood measles. Two large

doses (60,000 REQ each) of a water-soluble vitamin A

formulation given to children on each of two days

decreases the risk of death from measles 81% in areas of

prevalent vitamin A-deﬁciency.

FURTHER READING

Blomhoff, R., Green, M. H., Green, J. B., Berg, T., and Norum, K. R.

(1991). Vitamin A metabolism: new perspectives on absorption,

transport, and storage. Physiological Reviews 71, 951– 990.

Harrison, E. H. (1998). Lipases and carboxyesterases: possible roles in

the hepatic metabolism of retinol. Annual Reviews of Nutrition 18,

259–276.

Maden, M. (2001). Role of retinoic acid in embryonic and post-

embryonic development. Proceedings of the Nutrition Society

59, 65–73.

Napoli, J. L. (2000). Retinoic acid: its biosynthesis and metabolism.

Progress in Nucleic Acids Research 63, 139–188.

Newcomer, M. E. (1995). Retinoid-binding proteins: structural

determinants important for function. FASEB Journal 9, 229–239.

Saari, J. C. (2000). Biochemistry of visual pigment regeneration: the

Friedenwald lecture. Investigative Ophthalmology and Visual

Science 41, 337–348.

Stephensen, C. B. (2001). Vitamin A, infection, and immune function.

Annual Reviews of Nutrition 21, 167–192.

Sun, S. Y., and Lotan, R. (2002). Retinoids and their receptors in cancer

development and chemoprevention. Critical Reviews in Oncology

and Hematology 41, 41–55.

Wolf, G. (1984). Multiple functions of vitamin A. Physiological

Reviews 64, 873–937.

BIOGRAPHY

Joseph L. Napoli is a Professor in the Department of Nutrition Sciences

and Toxicology and a biochemist in the experimental station at UC-

Berkeley. His main research interests are in the metabolism and

functions of retinoids. He received his Ph.D. from the University of

Michigan-Ann Arbor in medicinal chemistry and was a post-doctoral

fellow in biochemistry at the University of Wisconsin-Madison.

VITAMIN A (RETINOIDS) 359

Vitamin B

and B

-Proteins

Bernhard Kra

utler

University of Innsbruck, Innsbruck, Austria

Vitamin B

(CNCbl), the antipernicious anemia factor, is

required for human and animal metabolism and was dis-

covered in the late 1940s. The B

-derivatives belong to the

tetrapyrrolic natural compounds and are cobalt complexes of

the unique and remarkably complex corrin ligand. The B

coenzymes are the cofactors in important organometallic

enzymatic reactions and are particularly relevant in the

metabolism of anaerobes. Indeed, the microorganisms are the

only natural sources of the B

-derivatives, whereas most

living organisms (except for the higher plants) depend on these

cobalt corrinoids. Vitamin B

and its derivatives thus hold an

important position in the life sciences and have attracted strong

interest from medicine, biology, chemistry, and physics.

: Structure and Reactivity

The red, cyanide-containing cobalt-complex vitamin B

(cyanocobalamin, CNCbl) is a relatively inert and

physiologically rather inactive Co(III)-corrin. The bio-

logically relevant B

-derivatives are the light sensitive

and chemically more labile organometallic coenzyme

forms, coenzyme B

-deoxy-5

-adenosylcobalamin,

AdoCbl), and methylcobalamin (MeCbl, see Figure 1).

STRUCTURE OF VITAMIN

-DERIVATIVES

The structures of vitamin B

(CNCbl) and of coenzyme

(AdoCbl) were established by X-ray crystallographic

studies from the laboratory of D. C. Hodgkin. This work

helped clarify the nature of the corrin ligand and to

discover the organometallic nature of the coenzyme

AdoCbl (see Figure 1). CNCbl and other cobalamins, in

which the cyanide ligand is replaced by another “upper”

-ligand (see Figure 1), represent the most common of

the nucleotide containing (i.e., “complete”) B

-deriva-

tives. The crystal structures of various (organometallic)

Co(III)-corrins were analyzed, including MeCbl, to

study the axial bonding at the corrin-bound cobalt

center and the inherent “non-planar” nature of the

corrin ligand (as opposed to the porphyrin ligand), as

well as possible implications of this for B

-catalyzed

enzymatic reactions. An interesting structure is that of

the oxygen-sensitive Co(II)-corrin cob(II)alamin (B

12r

the corrinoid moiety resulting from (Co–C)-bond

homolysis of AdoCbl during the catalytic cycle of

coenzyme B

-dependent enzymes.

UV/vis- and circular dichroism (CD)-spectroscopy

have been used to study the colored and chiral B

derivatives in solution. Nuclear magnetic resonance

(NMR) spectroscopy and mass spectrometry helped to

identify the corrinoids from anaerobes (methanogens,

sulfur metabolizing, and acetogenic bacteria) and to

characterize their solution structures. The Co(II)-forms,

in turn, have been investigated by electron spin

resonance (ESR) spectroscopy, a technique used increas-

ingly to analyze for paramagnetic intermediates in

-catalyzed enzymatic reactions.

The natural B

-derivatives are either “complete” or

“incomplete” corrinoids (which lack the nucleotide

function, see Figure 1). The natural “complete” corri-

noids carry different functional

-axial ligands. In

addition they may vary by the constitution of the

“nucleotide base,” a 5,6-dimethylbenzimidazole (DMB)

in the cobalamins (such as CNCbl), but an adenine in

pseudovitamin B

. The “complete” corrinoids are also

unique due to the unusual

-conﬁguration of their

(pseudo-)nucleotide appendage. The speciﬁc build-up of

this function enables the heterocyclic base to bind in an

intramolecular fashion to the “lower”

-axial coordi-

nation site of the corrin-bound cobalt center. In this way,

the nucleotide function steers the organometallic reac-

tivity at the cobalt center of “complete” B

-derivatives

and is also relevant for recognition and tight binding by

the B

-binding proteins.

-DERIVATIVES IN ELECTRON

TRANSFER REACTIONS

Oxidation– reduction processes are of key importance

for the chemistry and biology of B

. Under physiologi-

cal conditions B

-derivatives may exist in three

different oxidation states (Co(III), Co(II), or Co(I)),

each possessing different coordination properties and

reactivities: Axial coordination to the corrin-bound

cobalt center depends primarily on the formal oxidation

state of the cobalt ion. As a rule, the number of axial

ligands decreases in parallel with the cobalt oxidation

state: In the thermodynamically predominating forms of

cobalt corrins, two axial ligands are bound to the

diamagnetic Co(III)-center, one axial ligand is bound to

the paramagnetic Co(II)-center and axial ligands are

assumed to be absent at the diamagnetic Co(I)-center.

Electron transfer reactions involving B

-derivatives,

therefore, are accompanied by changes of the number of

axial ligands and depend upon the nature of axial

ligands. Axial coordination of the nucleotide base and of

strongly coordinating ligands stabilizes the cobalt center

against reduction and the reduction of alkyl-Co(III)-

corrins typically occurs at potentials more negative than

that of the Co(II)/Co(I)-redox-pair B

12r

12s

ORGANOMETALLIC REACTIONS

-DERIVATIVES

The reactivity of B

-derivatives in organometallic

reactions holds the key to much of the biological activity

of the B

-dependent enzymes: formation and cleavage

of the (Co–C)-bond in the B

-cofactors are essential

steps of the reactions catalyzed by B

-dependent

enzymes and are of particular interest.

In solution formation and cleavage of the (Co–C)-

bond in organometallic B

-derivatives were observed to

occur on all oxidation levels of the cobalt center. Two of

these reaction modes were also found to be relevant for

-dependent enzymatic reactions:

1. the homolytic mode of formation/cleavage of the

organometallic axial bond at the cobalt center (formally

a one-electron reduction/oxidation of the metal center,

see Figure 2), is of particular importance for the role of

AdoCbl as a cofactor AdoCbl is considered a “reversible

carrier of an alkyl radical” (or a reversibly functioning

“radical source”). The (Co–C)-bond of AdoCbl has

been determined to be , 30 kcal mol

strong and to be

affected only slightly by the nucleotide (in the “base-on”

form). The (reverse) reactions of B

12r

with alkyl

radicals (such as the 5

-deoxy-5

-adenosyl radical) are

remarkably fast. Indeed, the radicaloid B

12r

is a highly

efﬁcient “radical trap” and its reactions with radicals

can occur with minimal restructuring of the cobalt-

corrin moiety.

2. the heterolytic, nucleophile induced (SN

) mode of

formation/cleavage of the (Co–C)-bond at the cobalt

center (formally a two-electron oxidation/reduction of

the metal ion) is accompanied by formation/cleavage of

a second axial bond (Figure 2): Heterolytic formation/

cleavage of the (Co–C)-bond is particularly impor-

tant in enzyme-catalyzed methyl-transfer reactions

(Figure 3). This mode is represented by the reaction of

FIGURE 1 Left: structural formulas of vitamin B

(R ¼ CN, CNCbl), of coenzyme B

(R ¼ 5

-deoxy-5

-adenosyl, AdoCbl), methylcobalamin

(R ¼ methyl, MeCbl), cob(II)alamin (R ¼ e

12r

). Right/top: structure of coenzyme B

(AdoCbl). Bottom: symbols used for vitamin B

(R ¼ CN: CNCbl) and other cobalamins.

VITAMIN B

AND B

-PROTEINS 361

(“supernucleophilic”) Co(I)-corrins with alkylating

agents and by the nucleophile-induced demethylation

of methyl-Co(III)-corrins. Alkylation at the Co(I)-center

usually occurs via “classical” bimolecular nucleophilic

substitution (SN

). The intramolecular coordination of

the DMB-base in MeCbl has a notable thermodynamic

effect on this type of reaction: e.g., it stabilizes “base-

on” MeCbl by , 4 kcal mol

FIGURE 2 Elementary formal reaction steps of “complete” corrinoids characterizing their patterns of reactivity relevant for their cofactor

function in B

-dependent enzymes.

FIGURE 3 Biosynthesis of methionine by methylation of homocysteine is catalyzed by methionine synthase (MetH, Enz signiﬁes the MetH-

apoenzyme), where the bound corrinoid shuttles between MeCbl, in a “base-off/His-on” form, and cob(I)alamin.

362 VITAMIN B

AND B

-PROTEINS