The two head domains of the budding yeast Kinesin-14 Kar3Vik1 bind adjacent protofilaments at the start of the motility cycle, followed by release of Vik1 from one protofilament to allow the motor’s powerstroke.

Mammalian KIF3AC is classified as a heterotrimeric kinesin-2 that is best known for organelle transport in neurons, yet in vitro studies to characterize its single molecule behavior are lacking. The results presented show that a KIF3AC motor that includes the native helix α7 sequence for coiled-coil formation is highly processive with run lengths of ∼1.23 μm and matching those exhibited by conventional kinesin-1. This result was unexpected because KIF3AC exhibits the canonical kinesin-2 neck-linker sequence that has been reported to be responsible for shorter run lengths observed for another heterotrimeric kinesin-2, KIF3AB. However, KIF3AB with its native neck linker and helix α7 is also highly processive with run lengths of ∼1.62 μm and exceeding those of KIF3AC and kinesin-1. Loop L11, a component of the microtubule-motor interface and implicated in activating ADP release upon microtubule collision, is significantly extended in KIF3C as compared with other kinesins. A KIF3AC encoding a truncation in KIF3C loop L11 (KIF3ACΔL11) exhibited longer run lengths at ∼1.55 μm than wild-type KIF3AC and were more similar to KIF3AB run lengths, suggesting that L11 also contributes to tuning motor processivity. The steady-state ATPase results show that shortening L11 does not alter kcat, consistent with the observation that single molecule velocities are not affected by this truncation. However, shortening loop L11 of KIF3C significantly increases the microtubule affinity of KIF3ACΔL11, revealing another structural and mechanistic property that can modulate processivity. The results presented provide new, to our knowledge, insights to understand structure-function relationships governing processivity and a better understanding of the potential of KIF3AC for long-distance transport in neurons.

ABSTRACTMalonyl coenzyme A (malonyl-CoA) and methylmalonyl-CoA are two of the most commonly used extender units for polyketide biosynthesis and are utilized to synthesize a vast array of pharmaceutically relevant products with antibacterial, antiparasitic, anticholesterol, anticancer, antifungal, and immunosuppressive properties. Heterologous hosts used for polyketide production such asEscherichia colioften do not produce significant amounts of methylmalonyl-CoA, however, requiring the introduction of other pathways for the generation of this important building block. Recently, the bacterial malonyl-CoA synthetase class of enzymes has been utilized to generate malonyl-CoA and methylmalonyl-CoA directly from malonate and methylmalonate. We demonstrate that in the purple photosynthetic bacteriumRhodopseudomonas palustris, MatB (RpMatB) acts as a methylmalonyl-CoA synthetase and is required for growth on methylmalonate. We report theapo(1.7-Å resolution) and ATP-bound (2.0-Å resolution) structure and kinetic analysis ofRpMatB, which shows similar activities for both malonate and methylmalonate, making it an ideal enzyme for heterologous polyketide biosynthesis. Additionally, rational, structure-based mutagenesis of the active site ofRpMatB led to substantially higher activity with ethylmalonate and butylmalonate, demonstrating that this enzyme is a prime target for expanded substrate specificity.

Background: KIF3AB is a heterotrimeric plus-end-directed kinesin-2 motor, implicated in intraflagellar transport. Results: KIF3AB shows rate-limiting ADP release upon microtubule collision and an unusual apparent weak ATP affinity observed at microtubule⅐KIF3AB dissociation. Conclusion:The presteady-state kinetics suggest a novel ATPase mechanism for KIF3AB stepping. Significance: The KIF3AB kinetics reveal the mechanistic diversity that kinesin motors exhibit for cargo transport.

Background: Gcn5-related N-acetyltransferases (GNATs) modify proteins in all domains of life. Results: The structure of a GNAT was determined in complex with a protein substrate. Conclusion: Specificity of the GNAT-protein interaction is dictated by an extensive interaction surface compared with GNATpeptide structures. Significance: This is the first structure of a GNAT-protein acetylation complex, and it may enable structure-based identification and engineering of GNAT substrates.