Active Site Mutants of Escherichia coli Dethiobiotin Synthetase:  Effects of Mutations on Enzyme Catalytic and Structural Properties

Abstract

Five active site residues, Thr11, Glu12, Lys15, Lys37, and Ser41, implicated by the protein crystal structure studies of Escherichia coli DTBS, were mutated to determine their function in catalysis and substrate binding. Nine mutant enzymes, T11V, E12A, E12D, K15Q, K37L, K37Q, K37R, S41A, and S41C, were overproduced in an E. coli strain lacking a functional endogenous DTBS gene and purified to homogeneity. Replacement of Thr11 with valine resulted in a 24,000-fold increase in the Km(ATP) with little or no change in the Kd(ATP), KM(DAPA) and DTBS k(cat), suggesting an essential role for this residue in the steady-state affinity for ATP. The two Glu12 mutants showed essentially wild-type DTBS activity (slightly elevated k(cat)'s). Unlike wild-type DTBS, E12A had the same apparent KM(DAPA) at subsaturating and saturating ATP concentrations, indicating a possible role for Glu12 in the binding synergy between DAPA and ATP. The mutations in Lys15 and Lys37 resulted in loss of catalytic activity (0.01% and <0.9% of wild-type DTBS k(cat) for K15Q and the Lys37 mutant enzymes, respectively) and higher KM's for both DAPA (40-fold and >100-fold higher than wild-type for the K15Q and Lys37 mutant enzymes, respectively) and ATP (1800-fold and >10-fold higher than wild-type for K15Q and the K37 mutant enzymes, respectively). These results strongly suggest that Lys15 and Lys37 are crucial to both catalysis and substrate binding. S41A and S41C had essentially the same k(cat) as wild-type and had moderate increases in the DAPA and ATP KM and Kd (ATP) values. Replacement of Ser41 with cysteine resulted in larger effects than replacement with alanine. These data suggest that the H-bond between N7 of DAPA and the Ser41 side chain is not very important for catalysis. The catalytic behavior of these mutant enzymes was also studied by pulse-chase experiments which produced results consistent with the steady-state kinetic analyses. X-ray crystallographic studies of four mutant enzymes, S41A, S41C, K37Q, and K37L, showed that the crystals were essentially isomorphous to that of the wild-type DTBS. The models of these mutant enzymes were well refined (1.9 -2.6 A) and showed good similarity to the wild-type enzyme (rmsd of C alpha atoms: 0.16-0.24 A). The crystal structure of S41C complexed with DAPA, Mn2+/Mg2+, and AMPPCP revealed a localized conformational change (rotations of side chains of Cys41 and Thr11) which can account for the changes in the kinetic parameters observed for S41C. The crystal structures of the Lys37 mutant enzymes showed that the positive charge of the side chain of Lys37 is indispensable. Mutations of Lys37 to either glutamine or leucine resulted in a shift of the metal ion (up to 0.5 A) together with side chains of other active site residues which could disrupt the subtle balance between the positive and negative charges in the active site. The conformational change of the phosphate binding loop (Gly8-X-X-X-X-X-Gly14-Lys15-Thr16) upon nucleotide binding observed previously [Huang, W., Jia, J., Gibson, K. J., Taylo...

The BioC O-Methyltransferase Catalyzes Methyl Esterification of Malonyl-Acyl Carrier Protein, an Essential Step in Biotin Synthesis

“…Indeed, the BioC k cat values of ϳ200 s Ϫ1 are modest, although not nearly as low as those of the , respectively. Hence, when compared with BioD (29) and BioB (17), BioC is an effective catalyst. A rationale for the disparity between the first and concluding enzymes of the biotin synthetic pathway is that BioC must have reasonable activity to effectively compete with the 3-ketoacyl-ACP synthases for malonyl-ACP.…”

Nucleotide triphosphate promiscuity in Mycobacterium tuberculosis dethiobiotin synthetase

“…From the available structural data, the NTP binding site is divided into two discrete subsites; the phosphate-binding region (also known as the P-loop or Walker A motif) and the nucleoside-binding pocket. In E. coli DTBS (EcDTBS), a conformational change involving the P-loop (Gly8-X-X-X-X-X-Gly14-Lys15-Th16) was observed following phosphate binding [16]. In contrast, a comparison of available MtDTBS structures revealed no homologous conformational change upon the binding of inorganic phosphate [15].…”

“…The reaction mechanism has been proposed to involve three discrete steps, which are (1) the formation of N7-carbamate, (2) the formation of the carbamic-phosphoric acid anhydride, and (3) the closure of the ureido ring with the release of inorganic phosphate [14]. The amino acid residues required for binding the DAPA substrate and subsequent catalysis have been well characterized, with X-ray crystal structures available for the enzymes from Escherichia coli, M. tuberculosis, Helicobacter pylori, and Francisella tularensis [15], [16], [17] and [18]. However, the molecular details of NTP binding are less well defined, especially for M. tuberculosis DTBS (MtDTBS) where there is currently no crystal structure of the enzyme in complex with NTP.…”

“…Previous attempts to characterize DTBS have been restricted by the lack of facile assays. DTBS activity was commonly measured with a spectrophotometric assay by monitoring ADP production through the coupling of pyruvate kinase and lactate dehydrogenase [19], [16], [19] and [16]. Using two additional enzymes in the reaction is less than desirable for high-throughput screening applications.…”

Snapshot of a phosphorylated substrate intermediate by kinetic crystallography

“…One of the carbamate oxygens is within hydrogen bonding distance of the side chain of Lys-37. A crucial role of this residue in catalysis and substrate binding had been suggested by mutagenesis studies (23). The phosphoryl oxygens of the mixed carbamic-phosphoric acid anhydride intermediate interact through hydrogen bonds with main chain amides of Gly-118 and the side chains of Lys-15, Thr-11, Lys-37, and Glu-115, in addition to the site I Mg 2ϩ ion (Fig.…”