Tài liệu Báo cáo khoa học: Mechanism of dihydroneopterin aldolase NMR, equilibrium and transient

Mechanism of dihydroneopterin aldolase

NMR, equilibrium and transient kinetic studies of the

Staphylococcus aureus and Escherichia coli enzymes

Yi Wang, Yue Li, Yan Wu and Honggao Yan

Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI, USA

Dihydroneopterin aldolase (DHNA, EC 4.1.2.25)

catalyzes the conversion of 7,8-dihydro-d-neopterin

(DHNP) into 6-hydroxymethyl-7,8-dihydropterin (HP)

in the folate biosynthetic pathway, one of the principal

targets for developing antimicrobial agents [1]. Folate

cofactors are essential for life [2]. Most micro-organ￾isms must synthesize folates de novo. In contrast, mam￾mals cannot synthesize folates because of the lack of

three enzymes in the middle of the folate pathway, and

they therefore obtain folates from the diet. DHNA is

the first of the three enzymes that are absent in mam￾mals and therefore an attractive target for developing

antimicrobial agents [3].

DHNA is a unique aldolase in two respects. First, it

requires neither the formation of a Schiff’s base

between the substrate and enzyme nor metal ions for

catalysis [4]. Aldolases can be divided into two classes

based on their catalytic mechanisms [5,6]. Class I aldo￾lases require the formation of a Schiff’s base between

an amino group of the enzyme and the carbonyl of the

substrate, whereas class II aldolases require a Zn2+ ion

at their active sites for catalysis. The proposed catalytic

mechanism for DHNA [4,7,8] is similar to that of

class I aldolases, but the Schiff’s base is embedded in

the substrate (Fig. 1). Secondly, in addition to the aldo￾lase reaction, DHNA also catalyzes the epimerization

Keywords

dihydroneopterin aldolase; Escherichia coli;

folate biosynthesis; mechanism;

Staphylococcus aureus

Correspondence

H. Yan, Department of Biochemistry and

Molecular Biology, Michigan State

University, East Lansing, MI 48824, USA

Fax: +1 517 353 9334

Tel: +1 517 353 5282

E-mail: [email protected]

Website: http://www.bch.msu.edu/faculty/

yan.htm

*These authors have contributed equally to

this work

(Received 13 January 2007, revised 14

February 2007, accepted 28 February 2007)

doi:10.1111/j.1742-4658.2007.05761.x

Dihydroneopterin aldolase (DHNA) catalyzes both the cleavage of

7,8-dihydro-d-neopterin (DHNP) to form 6-hydroxymethyl-7,8-dihydro￾pterin (HP) and glycolaldehyde and the epimerization of DHNP to form

7,8-dihydro-l-monapterin (DHMP). Whether the epimerization reaction

uses the same reaction intermediate as the aldol reaction or the deprotona￾tion and reprotonation of C2¢ of DHNP has been investigated by NMR

analysis of the reaction products in a D2O solvent. No deuteration of C2¢

was observed for the newly formed DHMP. This result strongly suggests

that the epimerization reaction uses the same reaction intermediate as

the aldol reaction. In contrast with an earlier observation, the DHNA￾catalyzed reaction is reversible, which also supports a nonstereospecific

retroaldol ⁄ aldol mechanism for the epimerization reaction. The binding

and catalytic properties of DHNAs from both Staphylococcus aureus

(SaDHNA) and Escherichia coli (EcDHNA) were determined by equilib￾rium binding and transient kinetic studies. A complete set of kinetic con￾stants for both the aldol and epimerization reactions according to a unified

kinetic mechanism was determined for both SaDHNA and EcDHNA. The

results show that the two enzymes have significantly different binding and

catalytic properties, in accordance with the significant sequence differences

between them.

Abbreviations

DHMP, 7,8-dihydro-L-monapterin; DHNA, dihydroneopterin aldolase; DHNP, 7,8-dihydro-D-neopterin; EcDHNA, E. coli dihydroneopterin

aldolase; GA, glycolaldehyde; HP, 6-hydroxymethyl-7,8-dihydropterin; HPO, 6-hydroxymethylpterin; MP, L-monapterin; NP, D-neopterin;

SaDHNA, S. aureus dihydroneopterin aldolase.

2240 FEBS Journal 274 (2007) 2240–2252 ª 2007 The Authors Journal compilation ª 2007 FEBS