Crystal structures of two bacterial 3-hydroxy-3-methylglutaryl-CoA lyases suggest a common catalytic mechanism among a family of TIM barrel metalloenzymes cleaving carbon-carbon bonds.
The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) lyase catalyzes the terminal steps in ketone body generation and leucine degradation. Mutations in this enzyme cause a human autosomal recessive disorder called primary metabolic aciduria, which typically kills victims because of an inability to tolerate hypoglycemia. Here we present crystal structures of the HMG-CoA lyases from Bacillus subtilis and Brucella melitensis at 2.7 and 2.3 A resolution, respectively. These enzymes share greater than 45% sequence identity with the human orthologue. Although the enzyme has the anticipated triose-phosphate isomerase (TIM) barrel fold, the catalytic center contains a divalent cation-binding site formed by a cluster of invariant residues that cap the core of the barrel, contrary to the predictions of homology models. Surprisingly, the residues forming this cation-binding site and most of their interaction partners are shared with three other TIM barrel enzymes that catalyze diverse carbon-carbon bond cleavage reactions believed to proceed through enolate intermediates (4-hydroxy-2-ketovalerate aldolase, 2-isopropylmalate synthase, and transcarboxylase 5S). We propose the name "DRE-TIM metallolyases" for this newly identified enzyme family likely to employ a common catalytic reaction mechanism involving an invariant Asp-Arg-Glu (DRE) triplet. The Asp ligates the divalent cation, while the Arg probably stabilizes charge accumulation in the enolate intermediate, and the Glu maintains the precise structural alignment of the Asp and Arg. We propose a detailed model for the catalytic reaction mechanism of HMG-CoA lyase based on the examination of previously reported product complexes of other DRE-TIM metallolyases and induced fit substrate docking studies conducted using the crystal structure of human HMG-CoA lyase (reported in the accompanying paper by Fu, et al. (2006) J. Biol. Chem. 281, 7526-7532). Our model is consistent with extensive mutagenesis results and can guide subsequent studies directed at definitive experimental elucidation of this enzyme's reaction mechanism.
Forouhar
Farhad
F
Department of Biological Sciences and Northeast Structural Genomics Consortium, Columbia University, New York, NY 10027, USA.
Hussain
Munif
M
Farid
Ramy
R
Benach
Jordi
J
Abashidze
Mariam
M
Edstrom
William C
WC
Vorobiev
Sergey M
SM
Xiao
Rong
R
Acton
Thomas B
TB
Fu
Zhuji
Z
Kim
Jung-Ja P
JJ
Miziorko
Henry M
HM
Montelione
Gaetano T
GT
Hunt
John F
JF
D015079:2-Isopropylmalate Synthase / Q000737:chemistry; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry; D001412:Bacillus subtilis / Q000201:enzymology; D001665:Binding Sites; D017347:Brucella melitensis / Q000201:enzymology; D002244:Carbon / Q000737:chemistry; D002384:Catalysis; D020134:Catalytic Domain; D002412:Cations; D002850:Chromatography, Gel; D018360:Crystallography, X-Ray; D006801:Humans; D007700:Kinetics; D008027:Light; D008239:Lysine / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018384:Oxidative Stress; D007652:Oxo-Acid-Lyases / Q000737:chemistry*; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012542:Scattering, Radiation; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism
D002412:Cations; D001224:Aspartic Acid; D002244:Carbon; D015079:2-Isopropylmalate Synthase; C038482:4-hydroxy-2-ketovalerate aldolase; D007652:Oxo-Acid-Lyases; C021410:3-hydroxy-3-methylglutaryl-coenzyme A lyase; D008239:Lysine