Tseng Claire C, McLoughlin Shaun M, Kelleher Neil L, Walsh Christopher T
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA.
Biochemistry. 2004 Feb 3;43(4):970-80. doi: 10.1021/bi035714b.
DpgA is a bacterial type III polyketide synthase (PKS) that decarboxylates and condenses four malonyl-CoA molecules to produce 3,5-dihydroxyphenylacetyl-CoA (DPA-CoA) in the biosynthetic pathway to 3,5-dihydroxyphenylglycine, a key nonproteinogenic residue in the vancomycin family of antibiotics. DpgA has the conserved catalytic triad of Cys/His/Asn typical of type III PKS enzymes, and has been assumed to use Cys160 as the catalytic nucleophile to create a series of elongating acyl-S-enzyme intermediates prior to the C(8) to C(3) cyclization step. Incubation of purified DpgA with [(14)C]-malonyl-CoA followed by acid quench during turnover leads to accumulation of 10-15% of the DpgA molecules covalently acylated. Mutation of the active site Cys160 to Ala abrogated detectable covalent acylation, but the C160A mutant retained 50% of the V(max) for DPA-CoA formation, with a k(cat) still at 0.5 catalytic turnovers/min. For comparison, a C190A mutant retained wild-type activity, while the H296A mutant, in which the side chain of the presumed catalytic His is removed, had a 6-fold drop in k(cat). During turnover, purified DpgA produced 1.2 equivalents of acetyl-CoA for each DPA-CoA, indicating 23% uncoupled decarboxylation competing with condensative C-C coupling. The C160A mutant showed an increased partition ratio for malonyl-CoA decarboxylation to acetyl-CoA vs condensation to DPA-CoA, reflecting more uncoupling in the mutant enzyme. The Cys-to-Ala mutant thus shows the unexpected result that, when the normal acyl-S-enzyme mechanism for this type III PKS elongation/cyclization catalyst is removed, it can still carry out the regioselective construction of the eight-carbon DPA-CoA skeleton with surprising efficiency.
DpgA是一种细菌III型聚酮合酶（PKS），在3,5-二羟基苯甘氨酸的生物合成途径中，它使四个丙二酰辅酶A分子脱羧并缩合，生成3,5-二羟基苯乙酰辅酶A（DPA-CoA），3,5-二羟基苯甘氨酸是万古霉素类抗生素中的一种关键非蛋白质ogenic残基。DpgA具有III型PKS酶典型的保守催化三联体Cys/His/Asn，并且假定使用Cys160作为催化亲核试剂，在C(8)到C(3)环化步骤之前生成一系列延长的酰基-S-酶中间体。用[(14)C]-丙二酰辅酶A孵育纯化的DpgA，然后在周转过程中用酸淬灭，导致10-15%的DpgA分子发生共价酰化积累。活性位点Cys160突变为Ala消除了可检测到的共价酰化，但C160A突变体保留了50%的DPA-CoA形成的V(max)，k(cat)仍为0.5催化周转/分钟。作为比较，C190A突变体保留了野生型活性，而H296A突变体（其中假定的催化His的侧链被去除）的k(cat)下降了6倍。在周转过程中，纯化的DpgA每产生1个DPA-CoA就产生1.2当量的乙酰辅酶A，表明23%的未偶联脱羧与缩合性C-C偶联竞争。C160A突变体显示丙二酰辅酶A脱羧生成乙酰辅酶A与缩合生成DPA-CoA的分配比增加，反映出突变酶中更多的未偶联。因此，Cys-to-Ala突变体显示出意想不到的结果，即当去除这种III型PKS延长/环化催化剂的正常酰基-S-酶机制时，它仍然可以以惊人的效率进行八碳DPA-CoA骨架的区域选择性构建。
