Tài liệu Báo cáo khoa học: The crystal structure of coenzyme B12-dependent glycerol dehydratase in

The crystal structure of coenzyme B12-dependent glycerol dehydratase

in complex with cobalamin and propane-1,2-diol

Mamoru Yamanishi1

, Michio Yunoki1

, Takamasa Tobimatsu1

, Hideaki Sato1

, Junko Matsui1

, Ayako Dokiya1

,

Yasuhiro Iuchi1

, Kazunori Oe1

, Kyoko Suto2

, Naoki Shibata2

, Yukio Morimoto2

, Noritake Yasuoka2

and Tetsuo Toraya1

1

Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, Okayama; 2

Department of Life Science,

Himeji Institute of Technology, Hyogo, Japan

Recombinant glycerol dehydratase of Klebsiella pneumoniae

was purified to homogeneity. The subunit composition of

the enzyme was most probably a2b2c2. When (R)- and (S)-

propane-1,2-diols were used independently as substrates, the

rate with the (R)-enantiomer was 2.5 times faster than that

with the (S)-isomer. In contrast to diol dehydratase,an iso￾functional enzyme,the affinity of the enzyme for the (S)-

isomer was essentially the same or only slightly higher than

that for the (R)-isomer (Km(R)/Km(S) ¼ 1.5). The crystal

structure of glycerol dehydratase in complex with cyanoco￾balamin and propane-1,2-diol was determined at 2.1 A˚

resolution. The enzyme exists as a dimer of the abc hetero￾trimer. Cobalamin is bound at the interface between the a

and b subunits in the so-called
base-on mode with 5,6-

dimethylbenzimidazole of the nucleotide moiety coordina￾ting to the cobalt atom. The electron density of the cyano

group was almost unobservable,suggesting that the cyano￾cobalamin was reduced to cob(II)alamin by X-ray irradi￾ation. The active site is in a (b/a)8 barrel that was formed by a

central region of the a subunit. The substrate propane-1,2-

diol and essential cofactor K+ are bound inside the (b/a)8

barrel above the corrin ring of cobalamin. K+ is hepta￾coordinated by the two hydroxyls of the substrate and five

oxygen atoms from the active-site residues. These structural

features are quite similar to those of diol dehydratase. A

closer contact between the a and b subunits in glycerol

dehydratase may be reminiscent of the higher affinity of the

enzyme for adenosylcobalamin than that of diol dehydra￾tase. Although racemic propane-1,2-diol was used for cry￾stallization,the substrate bound to glycerol dehydratase was

assigned to the (R)-isomer. This is in clear contrast to diol

dehydratase and accounts for the difference between the two

enzymes in the susceptibility of suicide inactivation by gly￾cerol.

Keywords: coenzyme B12; adenosylcobalamin; glycerol

dehydratase; crystal structure; radical enzyme catalysis.

Adenosylcobalamin is one of the most unique compounds

in nature. It is a water-soluble organometallic compound

possessing a Co–C r bond and serves as a cofactor for

enzymatic radical reactions including carbon skeleton

rearrangements,heteroatom eliminations and intramolecu￾lar amino group migrations [1]. Diol dehydratase (EC

4.2.1.28) of Klebsiella oxytoca is an adenosylcobalamin

(AdoCbl1

) dependent enzyme that catalyzes the conversions

of 1,2-diols,such as propane-1,2-diol,glycerol,and 1,2-

ethanediol,to the corresponding aldehydes [2,3] (Fig. 1).

This enzyme has been studied intensively to establish the

mechanism of action of AdoCbl [4–7]. The structure–

function relationship of the coenzyme has also been

investigated extensively with this enzyme [5–8]. Recently,

we have reported the three-dimensional structures of its

complexes with cyanocobalamin [9] and adeninylpentylco￾balamin [10] and theoretical calculations of the entire energy

profile along the reaction pathway with a simplified model

[11–13]. In this sense,together with methylmalonyl-CoA

mutase [14],glutamate mutase [15],and class II ribonucleo￾tide reductase [16],diol dehydratase,is one of the most

suitable systems with which to study the structure-based

mechanisms of the AdoCbl-dependent enzymes [17,18].

Glycerol dehydratase (EC 4.2.1.30) catalyzes the same

reaction (Fig. 1) as diol dehydratase [19–21]. Although this

enzyme is isofunctional with diol dehydratase,these two

enzymes bear different physiological roles in the bacterial

metabolisms [6,7]. Selected genera of Enterobacteriaceae,

such as Klebsiella and Citrobacter,produce both glycerol

and diol dehydratases,but the genes for them are inde￾pendently regulated [22–25]: glycerol dehydratase is induced

when Klebsiella pneumoniae grows in the glycerol medium,

whereas diol dehydratase is fully induced when it grows in

the propane-1,2-diol-containing medium, but only slightly

in the glycerol medium. Glycerol dehydratase is a key

enzyme for the dihydroxyacetone (DHA) pathway

[23,26,27], and its genes are located in the DHA regulon

[28,29]. On the other hand, diol dehydratase is a key enzyme

for the anaerobic degradation of 1,2-diols [30,31], and its

genes are located in the pdu operon [32–34]. Furthermore,

although glycerol and diol enzymes are similar in their

Correspondence to T. Toraya,Department of Bioscience and

Biotechnology,Faculty of Engineering,Okayama University,

Tsushima-naka,Okayama 700–8530,Japan.

Fax: + 81 86 2518264,E-mail: [email protected]

Abbreviations: AdoCbl,adenosylcobalamin; aD, bD,and cD, a, b,and

c subunits of diol dehydratase; aG, bG,and cG, a, b,and c subunits of

glycerol dehydratase; buffer A,0.05 M potassium phosphate buffer

(pH 8); IPTG,isopropyl thio-b-D-galactoside.

(Received 11 June 2002,revised 23 July 2002,accepted 25 July 2002)

Eur. J. Biochem. 269,4484–4494 (2002)
FEBS 2002 doi:10.1046/j.1432-1033.2002.03151.x